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S M Downs

Publications and source records attributed to S M Downs.

70 records · Page 4Linked to original sources
PubMed1989

Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro.

Experiments were carried out to determine the specificity of growth factor action on maturation of the oocyte-cumulus cell complex in vitro. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with hypoxanthine (HX) and treated with one of ten different growth-promoting factors. The percentage of germinal vesicle breakdown (GVB) in the HX controls ranged from 44 to 64.7% after 21-22 h. Oocytes responded to treatment with growth-promoting factors in one of three ways: (1) no response; (2) low response; or (3) high response. The nonresponding groups included transforming growth factor-beta, platelet-derived growth factor, bombesin, sodium orthovanadate, nerve growth factor, and insulin-like growth factors I and II, each of which had no statistically significant effect on GVB. Insulin and fibroblast growth factor were members of the low response group and stimulated increases in GVB of 21.2 24.9%. Epidermal growth factor (EGF) was the only factor that produced a high frequency of maturation in the CEO; 100% of the arrested CEO were stimulated to undergo GVB in response to EGF treatment (a 51% increase over controls). No interaction was observed when EGF was tested with follicle-stimulating hormone (FSH) on hormone-induced GVB. When tested for an action on cumulus cell expansion, EGF was the only growth-promoting factor that triggered this response and did so more effectively than FSH. Heparin suppressed cumulus expansion in both EGF- and FSH-treated CEO, but did not prevent GVB stimulated by either hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin.

The efficacy of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)-containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP-supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte-cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP-dependent process. The frequency of FSH- and EGF-induced GVBD in cumulus cell-enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. of maturation is apparently not mediated solely by oocyte-cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.

Animals↗
PubMed1988

Gonadotropin-induced murine oocyte maturation in vivo is not associated with decreased cyclic adenosine monophosphate in the oocyte-cumulus cell complex.

The cyclic adenosine monophosphate (cAMP) content of intact oocyte-cumulus cell complexes at various times after the induction of oocyte maturation in mice in vivo was correlated with the time of commitment by the oocytes to undergo germinal vesicle breakdown (GVB) and metabolic coupling between the oocyte and cumulus cells. Seventy-nine percent of the oocytes either underwent GVB or were committed to do so by 2 h after injection of human chorionic gonadotropin (hCG). This occurred without a decrease in the coupling between cumulus cells and the oocyte and with increasing cAMP levels in the oocyte-cumulus cell complex. Maintenance of threshold levels of cAMP within mammalian oocytes appears essential for the maintenance of meiotic arrest, but data presented here suggest that oocyte maturation in mice is induced by gonadotropins in nonatretic follicles in vivo by some mechanism other than one which decreases the cAMP content of the intact oocyte-cumulus cell complex.

Animals↗
PubMed1987

The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation.

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2-4 mM, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod. 33, 1041-1049, 1985). In the present study, the effect of 2 mM hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 mM hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50-70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly (P less than 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function.

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.

Alkaline Phosphatase↗
PubMed1987

Induction of mouse oocyte maturation in vivo by perturbants of purine metabolism.

Previous studies have suggested a critical role for purines in the maintenance of mouse oocytes in meiotic arrest. If so, then disrupting specific purine metabolic pathways in vivo might induce the resumption of oocyte maturation. To test this hypothesis, immature mice were primed with pregnant mare's serum gonadotropin and 24 h later received, by i.p. injection, one of several drugs that inhibit specific enzymes in the purine metabolic pathways. Cumulus cell-enclosed oocytes were isolated from the ovaries at varying times after drug treatment and assessed for germinal vesicle breakdown (GVBD). The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid (MA) and bredinin (Br), each induced GVBD in a dose-dependent fashion, Br being the more effective agent. When the kinetics of oocyte maturation were examined, 71% of the oocytes from MA-treated mice had undergone GVBD 21-22 h after drug administration. Moreover, 100% GVBD was observed in oocytes from Br-treated mice after 6 h. The action of these drugs appeared to be a direct one and not mediated through stimulation of pituitary gonadotropin release or atresia. Azaserine, an inhibitor of de novo purine synthesis, also induced GVBD in a dose-dependent manner. However, sodium hadacidin and dl-alanosine, inhibitors of adenylosuccinate synthetase, failed to elicit a maturational response in oocytes in vivo. These data support an essential role for guanyl and/or xanthyl derivatives in the maintenance of meiotic arrest in vivo.

Animals↗
PubMed1986

Maintenance of murine oocyte meiotic arrest: uptake and metabolism of hypoxanthine and adenosine by cumulus cell-enclosed and denuded oocytes.

To analyze the potential mechanisms by which hypoxanthine and adenosine maintain meiotic arrest in mouse oocytes this study focused on: the uptake and metabolism of hypoxanthine and adenosine; the effect of inhibitors of inosine monophosphate (IMP) dehydrogenase on purine-mediated meiotic arrest; and the role of adenosine metabolism on the maintenance of meiotic arrest. Although the denuded oocyte can take up radiolabeled hypoxanthine and adenosine, an intact cumulus oophorus greatly augments uptake of these molecules (and/or metabolites). Both of these compounds were completely metabolized during incubation in vitro: hypoxanthine was apparently metabolized to uric acid and adenosine was metabolized to ADP; a small amount of each compound was also converted to inosine by cumulus cells and transferred to the oocyte. The IMP dehydrogenase inhibitors, bredinin and mycophenolic acid (MA), induced, in a dose-dependent manner, the resumption of maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine but had no effect on denuded oocytes. MA did not induce maturation when meiotic arrest was maintained by guanosine. Nor did MA alter the uptake of hypoxanthine by cumulus cell-enclosed oocytes. The poorly metabolized analog of adenosine, 2-chloroadenosine, was as effective as adenosine in its synergistic action with hypoxanthine in maintaining meiotic arrest. It is concluded that hypoxanthine and adenosine are metabolized within the oocyte-cumulus cell complex; xanthyl and/or guanyl compounds are produced by oocyte-cumulus cell complexes in the presence of hypoxanthine and play an important role in the maintenance of meiotic arrest; and adenosine need not be metabolized to act synergistically with hypoxanthine in maintaining meiotic arrest.

2-Chloroadenosine↗
PubMed1986

The role of purines in the maintenance of meiotic arrest in mouse oocytes.

Meiotic arrest of mammalian oocytes within ovarian follicles is maintained by a specific factor(s) within the follicle. There is strong evidence that cAMP plays an important role in the control of meiosis. Purines have also been implicated in the maintenance of meiotic arrest in vivo. Hypoxanthine and/or adenosine have been identified in pig and mouse follicular fluid and exert a meiosis-arresting action on mouse oocytes in culture. While adenosine apparently need not be metabolized to exert its action on oocyte maturation, the action of hypoxanthine is apparently due to the production of guanyl and/or xanthyl compounds by the oocyte-cumulus cell complex. The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid and bredinin, induced maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine. Hypoxanthine and adenosine are not toxic to oocytes, because oocytes undergo normal fertilization and pre- and post-implantation development following exposure to these molecules in vitro. It is not known how gonadotropins stimulate the resumption of meiosis within the follicle, but there are several possibilities: (1) the intrafollicular level of an oocyte maturation inhibitor is decreased; (2) the oocyte is uncoupled from surrounding follicle cells; (3) an inhibitory molecule is secreted or metabolized by the oocyte; and/or (4) a positive stimulus is produced by the follicle that overrides the presence of inhibitory molecules. Preliminary evidence suggests that cumulus cells may produce a positive stimulus that induces the maturation of cultured cumulus cell-enclosed oocytes. Whether germinal vesicle breakdown in vivo results from a positive induction, a loss of inhibitory input, or a combination of these two mechanisms remains to be determined.

Adenosine↗
PubMed1985

Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid.

Studies were carried out to identify and quantify the porcine follicular fluid (PFF) component(s) responsible for inhibition of murine oocyte maturation. A low molecular weight fraction of PFF (less than 1000) was prepared by dialysis and used in all experiments. This PFF fraction contained an inhibitor(s) of mouse oocyte maturation that absorbed maximally in the ultraviolet (UV) range at 250-260 nm. When the PFF fraction was charcoal-extracted, significant loss of absorbance at 250, 260, and 280 nm resulted, which corresponded to loss of inhibitory activity. Four major components of PFF were separated by ion-exchange chromatography and characterized according to their UV spectral characteristics and inhibitory activity. When individual fractions demonstrating identical spectra were pooled and analyzed by high-performance liquid chromatography, the first pooled fraction (A) was found to be impure, but adenine comprised 80% of the UV-absorbing material. Fractions B, C, and D were characterized as pure uracil, hypoxanthine, and 7-methylinosine, respectively. The concentrations of these compounds in PFF were estimated to be 0.06 mM adenine, 0.44 mM uracil, 1.41 mM hypoxanthine, and 0.19 mM 7-methylinosine. Comparison of the potencies of commercial preparations of these compounds established that hypoxanthine is the major inhibitory component of the low molecular weight PFF fraction. Moreover, a commercial preparation of hypoxanthine mimicked the action of PFF on mouse oocyte maturation in that it produced a transient inhibition of oocyte maturation that was significantly potentiated by follicle-stimulating hormone and dibutyryl cyclic adenosine monophosphate. When the inhibitory efficacies of purine and pyrimidine bases and nucleosides were compared, their relative potencies in decreasing order were purine bases greater than purine nucleosides greater than pyrimidine bases = pyrimidine nucleosides. We conclude that hypoxanthine is the predominant low molecular weight component of PFF that inhibits mouse oocyte maturation but that other purines/pyrimidines may also play a role in vivo in maintaining meiotic arrest.

Animals↗
PubMed1984

Chemical signals that regulate mammalian oocyte maturation.

The nature and functions of the chemical signals involved in the acquisition of competence to resume meiosis, and the maintenance of meiotic arrest in antral follicles are the subjects of this paper. Evidence indicating that gonadotropins are not required for the development of competence to undergo spontaneous maturation is discussed. However, gonadotropins may promote optimal conditions for oocyte development via an estrogen-dependent action on follicular cells. Evidence for the participation of cyclic AMP (cAMP), steroids and a putative maturation-inhibiting factor in the maintenance of meiotic arrest in mammalian oocytes is discussed. Cyclic AMP seems to play a critical role in the maintenance of meiotic arrest by actions in both granulosa/cumulus cells and oocytes. However, cAMP does not appear to equilibrate between cumulus cells and oocytes. In the granulosa/cumulus cells, cAMP may promote the generation/activation of a maturation-inhibiting factor which is transferred to the oocyte. Oocyte cAMP appears to be produced in the oocyte itself. The putative maturation-inhibiting factor may be maintained in an active form by a cAMP-dependent process in the oocyte. Alternatively, the putative maturation-inhibiting factor may play a role in maintaining oocyte cAMP levels. Some steroid hormones act synergistically with a cAMP-dependent process in the oocyte to maintain meiotic arrest. However, the physiological significance of this observation remains in question.

Animals↗
PubMed1984

Cyclic adenosine monophosphate and ovarian follicular fluid act synergistically to inhibit mouse oocyte maturation.

The relationship between cAMP and the capacity of two partially-purified fractions of porcine follicular fluid (PFF) to suppress mouse oocyte maturation was investigated. The fractions used were: 1) a low molecular weight filtrate (less than 10,000) derived from Amicon PM10 filtration of PFF (PM10), and 2) a fraction from Bio-Gel P2 chromatography of the PM10 filtrate (Bio-Gel) that has meiotic inhibitory activity in porcine oocytes. PM10 alone produced a transient inhibition of the maturation of both cumulus cell-enclosed and denuded mouse oocytes, as manifested by germinal vesicle breakdown. The addition of FSH or (Bu)2cAMP to medium containing either of the PFF fractions resulted in dramatic synergism of the inhibitory effect of PFF alone in cumulus cell-enclosed oocytes. Forskolin or (Bu)2cAMP promoted a similar synergistic response with either PFF fraction in denuded oocytes. The degree of inhibition was consistently greater in cumulus cell-enclosed than in denuded oocytes. The putative inhibitor was dialyzable through tubing having a nominal molecular weight cutoff of 1,000. Proteolysis, acid hydrolysis, or ether extraction of PM10 did not reduce its inhibitory synergism with (Bu)2cAMP. However, inhibition was completely abolished by charcoal extraction. Steroid hormones did not mimic the PM10-induced synergism when added to (Bu)2cAMP-containing medium in a concentration at least 14-fold greater than that present in PM10-supplemented medium. In conclusion, our results demonstrate the presence of a factor(s) in PFF that acts synergistically with a cAMP-dependent process to inhibit oocyte maturation in vitro. Furthermore, although PFF fractions suppressed the maturation of both denuded and cumulus cell-enclosed oocytes, cumulus cells appear to mediate the inhibitory activity of PFF. The data also suggest that the PFF inhibitor is a small (mol wt less than 1,000) hydrophobic molecule but not a peptide or nonpolar lipid.

1-Methyl-3-isobutylxanthine↗
PubMed1983

An ultrastructural study of preovulatory apical development in mouse ovarian follicles: effects of indomethacin.

The effects of the prostaglandin synthesis inhibitor, indomethacin, on the preovulatory morphology of apical follicle walls have been examined by transmission electron microscopy. Immature mice, superovulated with 5 IU pregnant mare serum (PMS) followed 40 hours later by 80 IU luteinizing hormone (LH) were treated with either 10 mg/kg indomethacin or an equivalent volume of the indomethacin vehicle 10 minutes prior to LH. Follicular apices from both groups were compared at 12 hours post-LH. Indomethacin treatment suppressed many of the morphological changes normally occurring in the apex during preovulatory development. Whereas apices from vehicle-treated animals demonstrated marked deterioration, dissociation, and thinning of tissue, the cell layers of apices from indomethacin-treated animals remained thickened and tightly packed, with limited signs of disruption. The results presented herein are consistent with the idea that prostaglandins are essential mediators of ovulation and suggest that these lipids augment apical rupture by mobilizing granulosa cells and stimulating the loss of connective tissue elements.

Animals↗
PubMed1983

Prostaglandins and preovulatory follicular maturation in mice.

Experiments have been carried out in an effort to reverse the indomethacin-induced inhibition of preovulatory follicular development in immature superovulated mice utilizing prostaglandins E2 and F2 alpha. All mice were primed with 5 IU pregnant mare's serum gonadotropin followed 40 h later by 80 IU luteinizing hormone (LH). Animals were sacrificed 10 1/2 or 11 1/2-12 h post-LH, at which time ovaries were fixed and prepared for microscopic observation. Control mice receiving both indomethacin and prostaglandin (PG) vehicles averaged 92% germinal vesicle breakdown, and 82% of maturing oocytes were surrounded by an expanded cumulus oophorus. Ovarian weight increased by 29% and the apical walls of preovulatory follicles demonstrated appreciable thinning following LH administration. In mice receiving indomethacin plus PG vehicle, follicular maturation was suppressed in a dose-dependent manner; in mice receiving 10 mg/kg, less than 50% of the oocytes resumed meiosis and, of these, only 9% were accompanied by cumulus expansion. Ovarian weight gain was also inhibited, and the apical follicle wall exhibited few signs of preovulatory thinning. PGE2 and PGF2 alpha both reversed the inhibition of cumulus and oocyte maturation induced by indomethacin, though PGE2 was more effective. Only PGF2 alpha promoted apical follicular thinning, and neither PG had a significant effect on ovarian weight. We conclude that, in mice, PGs may play an integral role during preovulatory maturation of the oocyte and cumulus, as well as thinning of the apical wall.

Animals↗
PubMed1982

Effects of indomethacin on preovulatory follicles in immature, superovulated mice.

In order to demonstrate the possible role of prostaglandins in preovulatory follicular development, immature mice superovulated with pregnant mare serum followed 40 hours later by luteinizing hormone (LH) were treated with the prostaglandin-synthetase inhibitor, indomethacin. Indomethacin (10 mg/kg) injected at varying intervals prior to or following LH inhibited ovulation most effectively when administered within 2 hours of the ovulatory gonadotropin. This inhibition was accompanied by (1) suppression of the morphological changes normally occurring within the follicular wall during preovulatory development and (2) failure of germinal vesicle breakdown (GVBD) in two-thirds of the follicles examined. When GVBD occurred, indomethacin treatment appeared to delay meiotic maturation. Cumulus tissue was more compact than in control follicles and maintained a close association with the oocyte. These results suggest that alterations in the morphology of the follicle prior to ovulation--specifically, thinning of the apical follicular wall and meiotic maturation--are regulated by prostaglandins.

Animals↗