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S M Downs

Publications and source records attributed to S M Downs.

At least 55 records · Page 3Linked to original sources
PubMed1995

The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte.

The effects of glucose and cumulus cells on oocyte ATP levels and germinal vesicle breakdown (GVB) in isolated mouse oocytes have been examined. Oocyte-cumulus cell complexes or denuded oocytes (DO) from pregnant mare serum gonadotropin-primed immature mice were cultured in minimum essential medium containing 4 mM hypoxanthine and 1 mM pyruvate, in the absence or presence of 0.55 mM glucose. After 17-18 hr in the presence of glucose, ATP in both the oocyte-cumulus cell complexes and oocytes derived from such complexes (cumulus cell-enclosed oocytes; CEO) was elevated, and less than one-half of the oocytes had resumed maturation (48% GVB). Removal of glucose caused a decrease in ATP levels in complexes and CEO and reversed the meiotic arrest in CEO (98% GVB), and these effects were mimicked by iodoacetate treatment. Higher frequencies of GVB were observed in glucose-free medium after more than 6 hr of culture, while ATP levels were reduced within 3 hr. Glucose had no effect on ATP levels or the meiotic state of denuded oocytes. Interestingly, iodoacetate had a stimulatory effect on GVB in DO (86% GVB compared to 57% in controls), but did not effect ATP levels. Glycerrhetinic acid, a gap junction uncoupler, completely suppressed oocyte-cumulus cell coupling in cultured complexes (0.15% coupling compared to 16.6% in controls) and reversed the inhibitory effect of glucose on oocyte maturation (91 and 95% GVB at 10 and 25 microM compared to 42% GVB in controls). This agent also prevented follicle-stimulating hormone-induced meiotic maturation in dibutyryl cAMP-arrested CEO. These results thus implicate mediation by gap junctions of both inhibitory and stimulatory signals from the cumulus cells. Comparison of ATP levels in spontaneously maturing CEO in vitro with those from oocytes maturing in vivo in response to human chorionic gonadotropin revealed that a decrease in oocyte ATP preceded or accompanied GVB in spontaneously maturing oocytes but not in those maturing in vivo. The results of this study indicate that glucose-derived elevation of oocyte ATP contributes to meiotic arrest in cumulus cell-enclosed oocytes that is dependent on patient gap junctions. Furthermore, a model for reinitiation of oocyte maturation is supported in which spontaneous GVB results from cessation or interruption of inhibitory influences, while ligand-provoked GVB is brought about by the generation of stimulatory signals that override inhibitory input.

Adenosine Triphosphate↗
PubMed1994

A decision analysis of streptokinase plus heparin as compared with heparin alone for deep-vein thrombosis.

BACKGROUND: It is uncertain whether patients with proximal deep-vein thrombosis should be treated with streptokinase followed by intravenous heparin or with intravenous heparin alone. Published reports indicate that streptokinase plus heparin increases the risk of bleeding, including central nervous system bleeding and death, but decreases the risk of postphlebitic syndrome. Previous recommendations regarding these treatments have not considered patients' preferences or the values they attach to the possible outcomes of therapy. METHODS: We used decision analysis to combine published estimates of the probabilities of various adverse outcomes of treatment (bleeding, pulmonary embolism, postphlebitic syndrome, and death) with the values patients placed on these outcomes. We questioned 36 patients about the values they attached to each outcome. Sixteen patients had had deep-vein thrombosis, and 20 had not. RESULTS: By the values they attached to the outcomes, all 36 patients indicated that they were unwilling to accept an increased risk of death to avoid postphlebitic syndrome. According to the decision analysis, heparin alone was the better treatment for all 36 patients. As compared with streptokinase plus heparin, heparin alone provided 29 days of additional life expectancy over the predicted life expectancy of 20 years. Although the difference between the two treatments was small, heparin alone remained the better treatment in sensitivity analyses that examined the reasonable ranges of probabilities of the clinical outcomes. CONCLUSIONS: The values patients placed on the outcomes of treatment for deep-vein thrombosis support the use of heparin alone over the combined use of streptokinase and heparin.

Cerebral Hemorrhage↗
PubMed1994

Induction of meiotic maturation in vivo in the mouse by IMP dehydrogenase inhibitors: effects on the developmental capacity of ova.

The present study was conducted to examine the effects of priming duration and inosine monophosphate (IMP) dehydrogenase inhibitors on the fertilization and pre- and postimplantation development of ova from immature mice. Mice were primed with equine chorionic gonadotropin (eCG) and 1 d later were treated with mycophenolic acid (MA) or mizoribine (Mz; also known as bredinin) or an equal volume of the vehicle. Two days after priming, mice received human chorionic gonadotropin (hCG) and were mated with fertile males. Some mice received hormone but were not mated. Ova were isolated from the oviducts 23-24 hr post-hCG and cultured in Whitten's medium. Some mice were not killed until 7 or 19 d postvaginal plug to determine the extent of implantation or development to term, respectively. In superovulated control mice, 94% of ova developed to two-cells and 82% of these progressed to the blastocyst stage. Nineteen of 23 mice (83%) had implantations (24/mouse) and 20 of 24 mice (83%) had term embryos (11/mouse). Induction of meiotic maturation with Mz or MA after 1 d of priming, followed 1 d later by hCG injection and mating, resulted in a significant loss of preimplantation developmental capacity (20-22% two-cells; 29-50% blastocysts). These numbers were similar whether or not the mice were mated, indicating that the development was parthenogenetic. In addition, Mz treatment reduced the number of mice with implantation sites (13 of 19, 68%), the number of implantations per mouse (10), the viability of the implantations, and development to term (four of 24, mice, 17%; one embryo/mouse). Thus, most of the implanted embryos were resorbed after implantation. This effect could be attributed, in part, to each of the following: (1) parthenogenetic activation; (2) aging of the metaphase II oocyte before insemination; and (3) resumption of meiotic maturation before the oocyte had reached its full developmental potential. Coincident gonadotropin injection reversed the loss of development brought about by IMP dehydrogenase inhibitors. These data show that induction of premature meiotic maturation by IMP dehydrogenase inhibitors results in a significant loss of developmental capacity and indicates that active purine metabolic pathways in situ maintain meiotic arrest and prevent premature meiotic maturation that would result in compromised development.

Animals↗
PubMed1994

The participation of energy substrates in the control of meiotic maturation in murine oocytes.

In this study we have examined the effects of various energy substrate combinations on both the maintenance of meiotic arrest and follicle-stimulating hormone (FSH)-induced germinal vesicle breakdown (GVB) in isolated mouse oocytes treated with 4 mM hypoxanthine (HX). When cumulus cell-enclosed oocytes were cultured in HX minus glucose, the addition of pyruvate dose-dependently sustained oocyte viability and induced GVB (from 25% GVB at 0 mM pyruvate to 98% GVB at 1 mM pyruvate). FSH had a limited stimulatory effect at all pyruvate concentrations. Oxaloacetate, but no other intermediates of the tricarboxylic acid (TCA) cycle, mimicked the action of pyruvate on viability and GVB. Lactate alone induced maturation in the absence of pyruvate but required NAD for this effect when the medium included pyruvate. Of two TCA cycle inhibitors tested, malonate had no effect on pyruvate-induced GVB, and fluoroacetate was inhibitory only at the highest concentration (10 mM). Pyruvate, oxaloacetate, and lactate also stimulated maturation in denuded oocytes. Adding glucose back to the medium produced an inhibitory effect on meiosis in cumulus cell-enclosed oocytes up to a concentration of 23 mM and was associated with increases in ATP. Exposure to the glycolytic inhibitor, iodoacetate, eliminated both the meiotic arrest and ATP production mediated by glucose. High concentrations of glucose (111 mM) proved stimulatory to oocyte maturation. Glucose had no effect on denuded oocytes. FSH-induced maturation was dependent upon the presence of glucose but was not causally related to increases in lactate production. In addition, raising the pyruvate concentration 10-fold did not overcome the meiotic block. Coculture of denuded oocytes with dissociated cumulus cells, but not intact oocyte-cumulus cell complexes, stimulated GVB in HX-containing medium when glucose was added. However, both the cumulus cells and the complexes produced an increase in pyruvate production, indicating that the effect of the dissociated cumulus cells was not due principally to secretion of this carbohydrate into the culture medium. This study has demonstrated the importance of energy substrates in the meiotic response of oocytes in culture. Evidence failed to support a role for increased TCA cycle activity in the stimulatory action of pyruvate. The inhibitory effect of glucose is the result of glycolytic activity that produces increased levels of ATP, but production of ATP and glycolytic intermediates does not appear to be the mechanism whereby glucose mediates a stimulatory action on meiosis, either in response to high glucose levels or FSH treatment. Other avenues of glucose metabolism or interaction may be responsible for this effect.

Adenosine Triphosphate↗
PubMed1994

High performance liquid chromatography analysis of hypoxanthine metabolism in mouse oocyte-cumulus cell complexes: effects of purine metabolic perturbants.

This study was undertaken to examine the metabolism of hypoxanthine by mouse oocyte-cumulus cell complexes. Complexes were isolated from immature mice 48 h after priming with 5 IU eCG and culture for 3 h in medium containing 14C-hypoxanthine in the absence or presence of one of three metabolic inhibitors: alanosine, mycophenolic acid, or 6-mercaptopurine. Tissue extracts from complexes were analyzed by HPLC using either a C18 reversed-phase column (for separation of purine bases and nucleosides) or an ion exchange column (for separation of nucleotides). Most of the hypoxanthine taken up by complexes was salvaged to inosine monophosphate (IMP) and then converted to nucleotides. Metabolism favored the synthesis of adenyl nucleotides over guanyl nucleotides. No evidence of metabolism to uric acid via xanthine oxidase was encountered, and metabolism to inosine via purine nucleoside phosphorylase was negligible. A similar pattern of hypoxanthine metabolism was observed in extracts of oocytes that had been denuded after the culture period. Addition of alanosine to the culture medium significantly reduced the synthesis of adenyl nucleotides in complexes and partially shunted metabolism in the direction of guanyl nucleotides. However, neither alanosine nor another inhibitor of adenylosuccinate synthetase, hadacidin, significantly influenced the meiotic arrest maintained by hypoxanthine. Mycophenolic acid eliminated conversion of IMP to guanyl nucleotides but did not appreciably affect metabolism to other nucleotides. 6-Mercaptopurine produced an increase in the hypoxanthine-containing peaks, which was consistent with suppression of purine salvage. These results demonstrate that hypoxanthine is readily salvaged by the murine oocyte-cumulus cell complex and that the inhibitor-induced changes in metabolism are consistent with the presumed mechanism of action of each inhibitor. In addition, whereas metabolism favors conversion of IMP to adenyl nucleotides, synthesis of adenyl nucleotides by this route during the culture period is apparently not required for hypoxanthine-maintained meiotic arrest in vitro.

Adenylosuccinate Synthase↗
PubMed1994

Hospital-acquired urinary tract infections in the pediatric patient: a prospective study.

To determine through a prospective study the characteristics of hospital-acquired urinary tract infections (HAUTI) in children, 525 children subjected to bladder catheterization during a hospital admission were identified through surveillance of 12,316 admissions during a 24-month period. Urine culture results were available for 296 (56.4%) of the catheterized patients. In addition 12 noncatheterized children with a documented HAUTI were identified. The clinical courses of all patients with a HAUTI were followed for at least 6 months after their last HAUTI during the study period. Forty-four patients, 1 week to 17 years of age, with 1 or more HAUTI during a hospital unit admission were identified. A total of 51 HAUTI occurred. Thirty-nine (76.5%) of the infections occurred in patients subjected to catheterization. Thirty-two (10.8%) of 296 catheterized patients developed a HAUTI. Forty-three (84.3%) of the 51 infections were single organism infections. One HAUTI was associated with a wound infection with the same organism and one with a concurrent bacteremia with the same organism. Relapses were seen after 4 HAUTI. One reinfection was identified. There were no deaths directly associated with a HAUTI. Hospitalized children subjected to urinary tract catheterization are at significant risk for HAUTI. Complications are infrequent and not life-threatening.

Adolescent↗
PubMed1993

Purine control of mouse oocyte maturation: evidence that nonmetabolized hypoxanthine maintains meiotic arrest.

Hypoxanthine is present in preparations of follicular fluid and has been shown to suppress the spontaneous meiotic maturation of mammalian oocytes in vitro. The present experiments examined the possible role of hypoxanthine metabolism in mediating this meiotic arrest. Four putative inhibitors of the enzyme, hypoxanthine phosphoribosyltransferase (HPRT), which metabolizes hypoxanthine to inosine monophosphate, were tested on lysates of oocyte-cumulus cell complexes. At a concentration of 1 mM, 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP) suppressed enzymatic activity by 86% and 98%, respectively, while 6-azauridine and 2,6-bis-(hydroxyamino)-9-beta-D-ribofuranosyl-purine had no effect. MPTF and 6-MP increased the inhibitory effect of hypoxanthine on germinal vesicle breakdown, but the other agents did not. The 2 active agents had similar effects on salvage activity and hypoxanthine-maintained meiotic arrest in denuded oocytes. Also, oocytes from XO mice were more sensitive to the meiosis-arresting action of hypoxanthine than oocytes from XX littermates, which have twice the HPRT activity. The actions of the HPRT inhibitors were not due to their conversion to nucleotides via HPRT and negative feedback on purine de novo synthesis, because azaserine and 6-methylmercaptopurine riboside, which are more potent inhibitors of de novo synthesis, had a stimulatory, rather than inhibitory, effect on hypoxanthine-arrested oocytes. Furthermore, several lines of evidence indicate that metabolism of hypoxanthine to xanthine and uric acid by xanthine oxidase does not mediate the inhibitory action of this purine base on meiotic maturation. The data therefore suggest that nonmetabolized hypoxanthine is responsible for the meiotic arrest observed, most likely through suppression of cAMP degradation.

Animals↗
PubMed1992

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.

Adenylate Cyclase Toxin↗
PubMed1992

Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes.

In this study, the effect of different energy sources used in Eagle's minimum essential medium on the meiotic maturation of mouse oocytes in culture was examined. The effects of glucose (5.5 mmol 1(-1)), pyruvate (0.23 mmol 1(-1)) and glutamine (2 mmol 1(-1)) in different combinations were tested on the maturation of denuded oocytes in the presence or absence of 300 mumol dibutyryl cAMP 1(-1) during 17-18 h of culture. In the absence of cyclic nucleotide, only oocytes from those groups containing pyruvate resumed maturation at a high frequency (99-100% germinal vesicle breakdown); all other combinations resulted in < or = 54% germinal vesicle breakdown. When dibutyryl cAMP was introduced, all pyruvate-containing groups exhibited maturation frequencies of about 50%, whereas maturation in all other groups was negligible (< or = 10% GVB). Pyruvate was also important for the maintenance of viability in denuded oocytes (> or = 86% viability in pyruvate-containing medium; < or = 35% viability in pyruvate-free groups). When cumulus cell-enclosed oocytes were cultured in medium without inhibitor, all combinations of energy substrates supported high frequencies of maturation (> or = 89% germinal vesicle breakdown) and viability (> or = 91%). The addition of dibutyryl cAMP resulted in inhibition of meiotic maturation (5-33% germinal vesicle breakdown) in all cultures except the pyruvate-alone group (97% germinal vesicle breakdown). Viability in cumulus cell-enclosed oocytes was greatest when two or more energy substrates were present in the medium. Follicle-stimulating hormone (FSH) produced a stimulation of meiotic maturation in all cultures of meiotically arrested cumulus cell-enclosed oocytes, but maximal induction of germinal vesicle breakdown was dependent upon D-glucose. Concanavalin A (ConA)-induced meiotic maturation was also dependent upon D-glucose. Uptake and metabolism of D-glucose by the cumulus cells is important in mediating the stimulatory effects of these ligands on oocyte maturation because (1) both FSH and ConA stimulated uptake of D-glucose and 2-deoxyglucose but not 3-O-methylglucose; (2) phloretin prevented the stimulatory action of FSH and ConA on germinal vesicle breakdown at a concentration that suppressed ligand-induced uptake of D-glucose; (3) 2-deoxyglucose, a hexose that suppresses glycolysis, prevented the induction of meiotic maturation by FSH and ConA and (4) D-mannose, a glycolysable sugar, was as effective as D-glucose in supporting the ligand effects.(ABSTRACT TRUNCATED AT 400 WORDS)

3-O-Methylglucose↗
PubMed1991

The meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the choice of macromolecular supplement is of critical importance when testing the hormone responsiveness of isolated cumulus cell-enclosed oocytes in culture.

Animals↗
PubMed1991

Management of infants at risk for occult bacteremia: a decision analysis.

Because febrile infants with no obvious source of bacterial infection may have bacteremia, and because bacteremia is difficult to diagnose on clinical grounds, we used decision analysis to evaluate whether such infants should be treated with antibiotics, tested further, or sent home. Using a simple decision tree, we found that the decision to give empiric antibiotic treatment is the decision of choice. The difference in quality-adjusted life expectancy between the "best" and "worst" decisions was only 11 days. However, this difference translated to prevention of death or permanent disability in 60 cases per 100,000 febrile children. Further, empiric treatment remained the best management alternative unless the probability of bacteremia was less than 1.4% (less than any published prevalence), or the efficacy of treatment was less than 21%. Our analysis demonstrated that a test with far greater sensitivity than leukocyte count or other tests currently in use is needed to justify testing rather than treating empirically. Further, an enormous patient population would be needed to find a difference of both clinical and statistical significance between treated and untreated patients in a controlled trial. In the absence of such trials, we recommend blood culture and empiric antibiotic treatment of all infants at risk for occult bacteremia.

Anti-Bacterial Agents↗
PubMed1991

Hypoxanthine-maintained two-cell block in mouse embryos: dependence on glucose and effect of hypoxanthine phosphoribosyltransferase inhibitors.

The culture conditions under which hypoxanthine maintains a two-cell block in preimplantation mouse embryos were assessed. Hypoxanthine prevented embryo development past the two-cell stage at concentrations as low as 30 nM, and this inhibitory activity required the presence of D-glucose. The action of hypoxanthine plus D-glucose was reversed by glutamine and higher lactate. D-mannose substituted for D-glucose in supporting the inhibitory action of hypoxanthine, but L-glucose, D-fructose, and 2-deoxyglucose were much less effective. Other purine derivatives such as inosine and adenosine, but not xanthosine or uric acid, also blocked development at the two-cell stage at a concentration of 30 microM, and guanosine was inhibitory at higher doses. Assays of hypoxanthine phosphoribosyltransferase (HPRT) activity in lysates of four-cell embryos determined that the drugs 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP), but not 6-azauridine (6-AzaU), prevented salvage of hypoxanthine. In addition, MPTF and 6-MP produced a significant two-cell block, which did not depend upon the presence of hypoxanthine or D-glucose; whereas 6-AzaU was without effect. When embryos were cultured 2 days in the presence or absence of D-glucose, hypoxanthine salvage was significantly reduced in lysates of four-cell embryos exposed to D-glucose. D-glucose had no effect when added directly to the assay mixture. These data demonstrate that the ability of hypoxanthine to block embryo development at the two-cell stage depends on the presence of D-glucose or other glycolyzable sugars and suggest that inhibition of the purine salvage pathway promotes the two-cell block.

Animals↗
PubMed1991

Metabolic coupling and ligand-stimulated meiotic maturation in the mouse oocyte-cumulus cell complex.

Cumulus cells are metabolically coupled to the mammalian oocyte via heterologous gap junctions. One function attributed to the gap junctional communications is the transfer of regulatory signals that direct the meiotic state of the oocyte. However, the precise role of these junctions in meiotic maturation is still unclear. The aim of this study was to test the hypothesis that meiotic resumption is induced by the transfer of a stimulatory signal(s) from the cumulus cells to the oocyte through the gap junctional coupling pathway. We have previously shown that the mitogenic lectin concanavalin A (Con A) induces oocyte maturation in isolated cumulus cell-enclosed oocytes (CEO) when meiotic arrest is maintained with a number of different inhibitory agents [Biol Reprod 1990; 42:413-423]. In the present study, Con A stimulated maturation in dibutyryl cAMP (dbcAMP)-arrested CEO but not in denuded oocytes cocultured with cumulus cells. Heptanol, a known gap junction uncoupler, effectively prevented Con A- and FSH-induced maturation of intact CEO and dramatically reduced metabolic coupling between cumulus cells and the oocyte. However, this alcohol had no effect on denuded oocytes (DO) or on dbcAMP-arrested CEO in the absence of stimulating ligand. Con A and FSH produced only a minimal loss of coupling. When the effects of heptanol were compared with those of the n-alkanols hexanol and decanol, the efficacies of these agents as suppressors of Con A-stimulated oocyte maturation was directly related to their relative abilities to suppress metabolic coupling.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohols↗
PubMed1990

Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes.

The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Maturation of the mouse oocyte-cumulus cell complex: stimulation by lectins.

We have used carbohydrate-binding proteins, or lectins, as tools to investigate the physiological phenomena associated with the preovulatory maturation of the oocyte-cumulus cell complex. Certain lectins are mitogens, and since other mitogenic agents such as growth factors are known to stimulate meiotic maturation and cumulus expansion, we tested the ability of lectins to provoke these physiological responses. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) and treated with one of eleven different lectins. With the exception of pokeweed mitogen (PWM), all of the mitogenic lectins tested were able to induce germinal vesicle breakdown (GVB) in meiotically arrested oocytes, and this action required the presence of the somatic cumulus cells; in fact, either there was no effect or maturation was suppressed when cumulus cell-free oocytes (denuded oocytes; DO) were treated with lectins. None of the nonmitogenic lectins stimulated meiotic maturation in either CEO or DO. The mitogenic lectin concanavalin A (Con A) also induced maturation in CEO when meiotic arrest was maintained with hypoxanthine, guanosine, or 3-isobutyl-1-methylxanthine. The kinetics of spontaneous oocyte maturation in inhibitor-free medium were not altered by Con A. Only the mitogenic lectins that induced meiotic maturation stimulated cumulus expansion, with Con A the most active lectin. The actions of Con A on the maturation of the oocyte-cumulus cell complex were inhibited by methyl-alpha-D-mannopyranoside as predicted by its sugar-binding specificity. These results demonstrate that (1) lectins can stimulate maturation of the mouse oocyte-cumulus cell complex; (2) mitogenicity is associated with the positive activity of the lectins; and (3) cumulus cells mediate the stimulatory action of lectins on oocyte maturation, while inhibition of GVB occurs at the oocyte level. These data support the idea that common signals mediate the mitogenic and maturation-inducing actions of lectins.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Stimulation of parthenogenesis in mouse ovarian follicles by inhibitors of inosine monophosphate dehydrogenase.

The effects of hormonal priming and inosine monophosphate (IMP) dehydrogenase inhibitors on the meiotic maturation and parthenogenetic activation of mouse oocytes were examined in this study. In the first series of experiments, unprimed mice or mice primed 24 h with equine chorionic gonadotropin (eCG) received injections of the IMP dehydrogenase inhibitors, bredinin (Br) or mycophenolic acid (MA), followed by histological examination at 24 h, 48 h, and 72 h after drug administration. In both treatment groups, oocytes from nonatretic antral follicles were stimulated to undergo germinal vesicle breakdown by 24 h and became parthenogenetically activated as manifested by pronuclear formation and early cleavage divisions. The parthenotes underwent degeneration by 72 h. In the second part of this study, the effects of priming and drug treatment on parthenogenetic activation and subsequent developmental potential in vitro were examined. Mice were primed with eCG, and 24 or 48 h later received injections of Br or MA. Cumulus cell-enclosed oocytes were isolated 21-22 h later and assessed for maturation; those having undergone germinal vesicle breakdown were cultured and subsequently examined for embryonic development. In mice primed for 24 h, but not 48 h, Br and MA stimulated a significant number of oocytes to resume maturation in vivo; these subsequently underwent activation and developed to blastocysts in vitro. In another series of experiments, germinal vesicle-stage oocytes were isolated from primed or unprimed mice and cultured in vitro to permit spontaneous meiotic maturation. Nine percent of mature ova from 24-h-primed mice developed to 2-cell parthenotes; activation in ova from unprimed and 48-h-primed mice was considerably lower. A time-course experiment demonstrated that the extent of parthenogenetic activation in vivo following Br treatment was related to the period of time between drug injection and isolation of ova, the optimal period being 12 h. Neither Br nor MA had a direct activating effect on the oocytes as evidenced by an inability to induce parthenogenesis in vitro. Simultaneous injection of hCG with either Br or MA stimulated ovulation and prevented the parthenogenetic response. These data are consistent with the idea that conditions within the follicle promote parthenogenetic activation when the oocyte matures in the absence of gonadotropin stimulation.

Animals↗
PubMed1989

Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity.

Hypoxanthine and adenosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.: Biology of Reproduction 33:1041-1049. 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis-arresting levels of cyclic adenosine monophosphate (cAMP) in the oocyte. Oocyte-cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell-enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The addition of adenosine to hypoxanthine-containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP-dependent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purine, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes. The second hypothesis tested in this study is that purines maintain meiosis-arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiesterase activity. In descending order of potency, 3-isobutyl-1-methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the meiotic arrest and cAMP accumulation mediated by follicle-stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis-arresting levels of cAMP.

1-Methyl-3-isobutylxanthine↗