Monte Carlo simulation of O(2) phi(4) field theory in three dimensions.

Using standard numerical Monte Carlo lattice methods, we study non-universal properties of the phase transition of three-dimensional straight phi(4) theory of a two-component real field phi=(phi(1),phi(2)) with O(2) symmetry. Specifically, we extract the renormalized values of /u and r/u(2) at the phase transition, where the continuum action of the theory is integral d(3)x[1/2/inverted Delta phi/(2) + 1 2r phi(2)+(u/4!)phi(4)]. These values have applications to calculating the phase-transition temperature of dilute or weakly interacting Bose gases (both relativistic and nonrelativistic). In passing, we also provide perturbative calculations of various O(a) lattice-spacing errors in three-dimensional O(N) scalar field theory, where a is the lattice spacing.

Slow polymerization of Mycobacterium tuberculosis FtsZ.

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation.

Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin and OH-propyl-beta-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these beta-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different beta-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the beta-cyclodextrins with cholesterol-SO4- to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The beta-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The beta-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, beta-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.

Temporal patterns of gene expression of G1-S cyclins and cdks during the first and second mitotic cell cycles in mouse embryos.

Cell-cycle progression in somatic cells is regulated by a family of cyclins and cyclindependent kinases (cdks) that form specific complexes as a function of cell-cycle progression. However, the transcript abundance of G1-S cyclins and cdks during the meiotic and mitotic cell cycles of mammalian embryos has not been previously reported. Using a reverse transcription-polymerase chain reaction (PCR) assay that detects changes in either mRNA abundance or polyadenylation state, we examined the relative levels of gene expression for the G1-S cyclins and cdks, as well as for p21, p27, and the retinoblastoma (Rb) gene in mouse oocytes, metaphase II-arrested eggs, and 1-2-cell embryos. The PCR products for cyclins D1, D3, and A, as well as cdk4, p21, and Rb, displayed similar levels in meiotically incompetent and competent oocytes, as well as in metaphase II-arrested eggs. The levels of PCR products for cyclin D2, p27, and two forms of cdk2 were similar in meiotically incompetent and competent oocytes but decreased during oocyte maturation. Finally, the level of PCR products for cyclin E and cdk2 gradually decreased during the progression from meiotically incompetent oocytes to metaphase II-arrested eggs. When the levels of PCR products for the G1-S regulatory genes were evaluated during the first and second mitotic cell cycles, four main patterns were found: 1) steady levels for cyclin A; 2) steady levels followed by a 2-3-fold increase during the G2 phase of the second mitotic cell cycle for cyclins D1, E, cdk2, and p21; 3) a transient increase during the S and/or G2 phases of the first mitotic cell cycle for p27, cyclin D3, and the two forms of cdk2; and 4) higher levels during the first cell cycle and then a decrease with lower levels during the second mitotic cell cycle for cyclin D2 and Rb. cdk4 expression displayed a combination of patterns 2 and 3. The increase in the amount of PCR product for the cdk4 gene during the first mitotic cell cycle was due to polyadenylation, whereas the increase in the amount of PCR product for cdk4, cdk2, and cyclins D1 and E in the second mitotic cell cycle was a product of activation of the embryonic genome.

Potential role of mitogen-activated protein kinase in pronuclear envelope assembly and disassembly following fertilization of mouse eggs.

Changes in the activities of the p34cdc2/cyclin B complex and mitogen-activated protein (MAP) kinase were analyzed after insemination of mouse eggs in vitro. Whereas histone H1 kinase activity (p34cdc2/cyclin B) fell to negligible levels by 90 min postinsemination, a decrease to negligible levels of myelin basic protein kinase activity (i.e., MAP kinase) was not observed until about 7 h postinsemination. The decrease in MAP kinase activity appeared to be linked to the prior decline in p34cdc2/cyclin B kinase activity, since inhibiting the fertilization-induced destruction of cyclin B by treating eggs with the microtubule inhibitor nocodazole prevented the decrease in each of these protein kinases; an intact spindle is required for cyclin destruction. Moreover, experimental elevation of MAP kinase activity by okadaic acid treatment under conditions that maintain negligible levels of p34cdc2/cyclin B kinase activity suggested that MAP kinase could be involved in pronuclear envelope dynamics. Specifically, preventing the fertilization-induced decrease in MAP kinase activity was correlated with inhibiting pronucleus formation, and elevating MAP kinase activity subsequent to pronucleus formation resulted in precocious pronuclear envelope breakdown prior to entry into M phase.

Differential effect of activators of protein kinase C on cytoskeletal changes in mouse and hamster eggs.

Treatment of metaphase II-arrested hamster eggs with activators of protein kinase C has been reported to promote resumption of the cell cycle, second polar body emission, and pronucleus formation (G.I. Gallicano, S.M. Schwarz, R.W. McGaughey, and D.G. Capco, 1993, Dev. Biol. 156, 94-106). In contrast, we have not observed these responses in mouse eggs obtained from CF-1 mice treated with these activators. In this report, we evaluated if this difference was due to differences in the technique used for PKC stimulation in the two different laboratories or due to species differences. Metaphase II-arrested hamster or mouse eggs were treated with phorbol diesters for 5 min or with a membrane-permeable diacylglycerol for 1 hr. Treatment of hamster eggs resulted in (1) the formation of "second polar body-like structures" commencing 5 min after treatment and reaching a maximum by 20-40 min; (2) a remarkable increase in the staining of filamentous actin in the region of these polar body-like structures; and (3) the disassembly of spindle microtubules. A reduction in cdc2/cyclin B1 kinase activity, as assessed by a decrease in H1 kinase activity, as well as progression from metaphase to anaphase were not observed. Treatment of mouse eggs from either CF-1 or CD-1 mice with these activators of PKC did not result in the formation of these polar body-like structures, did not cause an increase in filamentous actin, and did not result in a reduction in histone H1 kinase activity. This treatment, however, did induce disassembly of the spindle microtubules and the formation of multiple "pronucleus-like structures" that were more discernible in eggs from CD-1 mice. We conclude that the "apparent" activation of hamster eggs by activators of PKC is due to the effect of these agents on the cytoskeleton, which gives rise to structures that appear similar to polar bodies, but without any evidence of cell cycle resumption. The different responses seen in mouse and hamster eggs are mainly due to differences in the sensitivity of the cytoskeleton to rearrangements induced by these agents.

Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.

In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129-1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.

Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation.

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs.

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

Complete mouse egg activation in the absence of sperm by stimulation of an exogenous G protein-coupled receptor.

Sperm-induced egg activation has several features in common with signal transduction pathways mediated by ligand-receptor-effector systems. G proteins are postulated to act as signal transducers regulating the plethora of cellular responses constituting egg activation that occur in response to sperm. We report that acetylcholine treatment of mouse eggs expressing the human m1 muscarinic receptor, a G protein-coupled receptor, results in the full complement of events of egg activation, including ZP2 to ZP2f conversion, recruitment of maternal mRNAs, pronuclear formation with subsequent DNA synthesis, cleavage to the two-cell stage, and activation of the embryonic genome. The induction of a full complement of sperm-induced egg activation events by ACh in the absence of sperm strongly suggests that sperm-induced egg activation is mediated by egg G proteins.

Rabbit endosalpinx inhibits implantation in vitro.

OBJECTIVE: To evaluate the ability of rabbit endometrial or endosalpingeal cells to support implantation in vitro and to assess the effects of endosalpinx and endometrium-conditioned media (CM) on blastocyst-endometrial cell interaction. DESIGN: In one experiment, rabbit blastocysts were co-cultured in vitro with endometrial or endosalpingeal cells growing on Matrigel-coated plastic culture plates or Millicell-HA inserts. In a second experiment, rabbit blastocysts were co-cultured with endometrial cells in the presence of fresh medium or of endosalpinx- or endometrial-CM. After 48 or 72 hours attachment to the cell monolayer was evaluated. RESULTS: Blastocysts in co-culture attached to endometrial but not to endosalpingeal monolayers. The addition of CM from cultured endosalpinx significantly decreased embryo attachment to endometrial cells in culture. CONCLUSIONS: These findings in vitro agree with the observation that rabbit endosalpinx in vivo does not support embryo implantation and support the hypothesis that rabbit endosalpinx secretes a factor that prevent tubal implantation.

Rabbit endosalpinx suppresses ectopic implantation.

This study was undertaken to elucidate the mechanism underlying the absence of oviductal ectopic pregnancies in infraprimates. Endosalpingeal circumferential grafts were substituted for endometrium in a group of rabbits. The endosalpingeal grafts interfered with implantation as evidenced by four observations: (1) The nidation indices were lowered, (2) no implantations occurred on the grafted endosalpinx, (3) unattached blastocysts were found in animals grafted with endosalpinx, and (4) the implantation sites were significantly smaller in uterine horns containing endosalpingeal grafts. These findings suggest the presence of a factor in rabbit endosalpinx that actively suppresses ectopic implantation in the oviduct.

Effects of delayed transfer and treatment with oestrogen on the transport of microspheres by the rat oviduct.

Starch or dextran blue microspheres were transferred microsurgically to the infundibulum of the oviduct on Days 1, 2, or 3 of pregnancy of control and oestradiol-treated rats. The animals were killed a few hours to several days after transfer to assess the number and distribution of ova and microspheres in the tract. After transfer on Day 1 of pregnancy, microspheres and eggs crossed the ampullary-isthmic junction (AIJ) 18 h after ovulation. After transfer on Day 2 of pregnancy, more than 50% of microspheres were retained in the ampulla, indicating that the AIJ changes again 34 h after ovulation. Treatment with oestradiol did not advance the passage of eggs or microspheres across the AIJ but caused accelerated transport through the isthmus as soon as the eggs or microspheres reached this segment. Dextran blue microspheres were seen to move back and forth in the isthmus of control anaesthetized rats at a frequency of 5-6 times/min. Between 7 and 20 h after treatment with oestradiol the frequency of these movements was significantly augmented, indicating that increased frequency of contractions of the smooth muscle of the isthmus precedes and accompanies accelerated transport of ova through this segment.

Synthetic microspheres transferred to the rat oviduct on day 1 of pregnancy mimic the transport of native ova.

Microspheres of various materials and diameters were transferred microsurgically to the rat oviduct on Day 1 of pregnancy and autopsies were done at various times thereafter up to Day 10 to assess the recovery and segmental distribution of microspheres and eggs in the genital tract or the viability of embryos. The number and distribution of eggs in the treated and control sides after unilateral transfers were not different on Day 4 and 5 and the number of embryos implanted on Day 10 was not significantly affected after bilateral transfers. The segmental distribution of eggs and starch microspheres within the oviduct on Days 2, 3 or 4 showed that both are transported partly intermingled from ampulla to uterus. When microspheres of poly(DL-lactide-co-glycolide), starch, dextran or dextran blue were transferred, their distribution in the genital tract in the morning of Day 5 showed that poly(DL-lactide-co-glycolide) and dextran microspheres stayed longer in the oviduct while starch and dextran blue microspheres were transported to the uterus at the same time as the eggs. Transfer of starch microspheres of 40-60 microns to one oviduct and 180-200 microns in diameter to the opposite oviduct showed that distribution on one side was nearly identical to that of the other side from Days 2 to 5. We conclude that the behaviour of synthetic surrogate ova in rats differs from that in rabbits. The rat oviduct does not change the rate of transport of native eggs following transfer of synthetic surrogate ova.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of sperm migration, fertilization and implantation in rats by precoital intrauterine administration of gossypol or nonoxynol-9.

The effects of precoital intrauterine administration of Gossypol, Nonoxynol-9 or p-Aminobenzamidine on reproductive function were investigated in rats. The drugs were instilled in one uterine horn in the evening of proestrus prior to caging with a fertile male. The opposite side was similarly treated with the vehicle and served as control. Animals were killed in the morning following mating to assess the number of spermatozoa at the site of fertilization or on days 2-3 of pregnancy to determine the proportion of penetrated oocytes or on day 12 to count implanted embryos. Gossypol 0.05 to 5 mg or Nonoxynol-9 0.1 - 10 microliter reduced the number of spermatozoa reaching the ampullae and the number of penetrated oocytes in a dose-dependent fashion. At the highest dose both compounds prevented pregnancy exclusively in the treated side. p-Aminobenzamidine at a dose of 10 mg failed to affect the proportion of penetrated eggs found on days 2 or 3 of pregnancy. The results indicate that the rat bioassay is suitable to screen agents that can affect sperm function or viability since it can discriminate between drugs and doses and disclose differential effects upon sperm migration and fertilizing capacity.

Essential drugs for church-related rural health care

In Kenya the government is supporting a programme to improve the supply of essential drugs to church-related rural health institutions and to train the staffs of these bodies in the rational use of the drugs