Bovine peripheral blood MSCs chemotax towards inflammation and embryo implantation stimuli.

Mesenchymal stem cells (MSCs) have a great potential in regenerative medicine because of their multipotential and immunoregulatory capacities, while in early pregnancy they could participate in the immunotolerance of the mother towards the embryo. Peripheral blood constitutes an accessible source of MSCs. We successfully isolated peripheral blood MSC (pbMSCs) lines, with or without previous bone marrow mobilization. All pbMSCs lines obtained in both conditions presented classical MSC markers and properties, alkaline phosphatase activity and multipotent capacity to differentiate among adipogenic, osteogenic or chondrogenic lineages, and suppressed the proliferation of T cells. pbMSCs showed migratory capacity without cytokine stimulation while increasing their migration rate in the presence of inflammatory or embryo implantation stimuli. Interestingly, in contrast to MSCs derived from endometrial tissue, three pbMSCs lines also showed increased migration towards the IFN-τ implantation cytokine. Moreover, the secretome produced by an early implantation stage embryonic trophectoderm cell line showed a chemoattractant effect in pbMSCs. Our results suggest that circulating MSCs are present in the peripheral blood under healthy conditions. The fact that both the inflammation and implantation signals induced pbMSCs chemotaxis highlights MSC heterogeneity and suggests that their migratory capacity may differ according to their tissue of origin and would suggest the possible active recruitment of MSCs from bone marrow during pregnancy to repress the immune response to prevent the embryo rejection by the maternal organism.

Spectral and Kinetic Study of 3-Styrylquinoxalin-2(1 H)-ones Photoreduced by N-Phenylglycine and Amines.

The photoreduction by amines and N-phenylglycine, NPG, of six styrylquinoxalin-2(1 H)-ones derivatives substituted in the styryl moiety, R-SQ, was studied by using flash photolysis. The photoreaction is initiated via a single electron transfer from the electron donor (amines or NPG) to R-SQ excited triplet state, 3R-SQ*, with the formation of a triplet state radical ion pair or a charge transfer exciplex, 3[CRIP/CTE]. These species live longer than the respective 3R-SQ* and have very similar transient spectra. In the presence of NPG, these 3[CRIP/CTE] evolve on µs time scale to the respective hydrogenated radicals, R-SQH•, whose transient spectra and reaction rate constants with NPG are reported. The identity of these hydrogenated radicals was supported by the spectra obtained with the α-H donor triethylamine and previous pulse radiolysis studies in 2-propanol. Our findings allow proposing a radical chain reaction mechanism that explains the observed spectral behavior and rationalizes formation of the main product formed by binding of four PhNHCH2• derived from NPG decarboxylation.

Radical Ions of 3-Styryl-quinoxalin-2-one Derivatives Studied by Pulse Radiolysis in Organic Solvents.

The absorption-spectral and kinetic behaviors of radical ions and neutral hydrogenated radicals of seven 3-styryl-quinoxalin-2(1 H)-one (3-SQ) derivatives, one without substituents in the styryl moiety, four others with electron-donating (R = -CH3, -OCH3, and -N(CH3)2) or electron-withdrawing (R = -OCF3) substituents in the para position in their benzene ring, and remaining two with double methoxy substituents (-OCH3), however, at different positions (meta/para and ortho/meta) have been studied by UV-vis spectrophotometric pulse radiolysis in neat acetonitrile saturated with argon (Ar) and oxygen (O2) and in 2-propanol saturated with Ar, at room temperature. In acetonitrile solutions, the radical anions (4R-SQ•-) are characterized by two absorption maxima located at λmax = 470-490 nm and λmax = 510-540 nm, with the respective molar absorption coefficients ε470-490 = 8500-13 100 M-1 cm-1 and ε510-540 = 6100-10 300 M-1 cm-1, depending on the substituent (R). All 4R-SQ•- decay in acetonitrile via first-order kinetics, with the rate constants in the range (1.2-1.5) × 106 s-1. In 2-propanol solutions, they decay predominantly through protonation by the solvent, forming neutral hydrogenated radicals (4R-SQH•), which are characterized by weak absorption bands with λmax = 480-490 nm. Being oxygen-insensitive, the radical cations (4R-SQ•+) are characterized by a strong absorption with λmax = 450-630 nm, depending on the substituent (R). They are formed in a charge-transfer reaction between a radical cation derived from acetonitrile (ACN•+) and substituted 3-styryl-quinoxalin-2-one derivatives (4R-SQ) with a pseudo-first-order rate constant k = (2.7-4.7) × 105 s-1 measured in solutions containing 0.1 mM 4R-3-SQ. The Hammett equation plot gave a very small negative slope (ρ = -0.08), indicating a very weak influence of the substituents in the benzene ring on the rate of charge-transfer reaction. The decay of 4R-SQ•+ in Ar-saturated acetonitrile solutions occurs with a pseudo-first-order rate constant k = (1.6-6.2) × 104 s-1 and, in principle, is not affected by the presence of O2, suggesting charge-spin delocalization over the whole 3-SQ molecule. Most of the radiolytically generated transient spectra are reasonably well-reproduced by semiempirical PM3-ZINDO/S (for 4R-SQ•-) and density functional theory quantum mechanics calculations employing M06-2x hybrid functional together with the def2-TZVP basis set (for 4R-SQ•+).

Spectral and Kinetic Study of 3-Methylquinoxalin-2-ones Photoreduced by Amino Acids: N-Phenylglycine Radical Chain Reactions and N-Acetyltryptophan Decarboxylation.

Transient intermediates were identified in the photoreduction of 3-methylquinoxalin-2-one derivatives by N-phenylglycine, NPG, and N-acetyltryptophan, NAT. For both reductants it can be postulated a sequence of reaction comprising first a photoinduced single electron transfer followed by a proton transfer from the radical cation of the electron donor to the radical anion of the 3-methylquinoxalin-2-one giving rise to the reported products. The effect of the concentrations of NPG and the quinoxalin-2-one on the rate of photoconsumption of this last were quantified, and the lifetimes of the possible intermediates estimated. In the photoreduction by NAT, processes leading to the decarboxylation of NAT and radical adduct product compete with the expected SET from the indoyl N to the excited triplet of quinoxalin-2-ones as revealed by the detection of the deprotonated N-acetyltryptophan radical [NAT-H](•). This radical is formed almost instantly after the laser pulse and has a secondary delayed growth via a delayed proton transfer from the indoyl radical cation NAT(•+) to the quinoxalin-2-one radical anions. The decarboxylation of NAT that mimics C-terminus tryptophan in proteins is biologically relevant because might cause damages at cellular and the whole organism level. As far as we know this is the first report of a radical decarboxylation of N-acetyltryptophan leading to photoproducts.

Dual Emission of a Novel (P,N) Re(I) Complex: A Computational and Experimental Study on [P,N-{(C6H5)2(C5H4N)P}Re(CO)3Br].

The spectroscopic, electrochemical, and photophysical properties of the new complex [P,N-{(C6H5)2(C5H4N)P}Re(CO)3Br] are reported. The UV-vis spectrum in dichloromethane shows an absorption maximum centered at 315 nm and a shoulder at 350 nm. These absorption bands have been characterized to have MLCT character. Excitation at both wavelengths (maximum and shoulder) leads to an emission band centered at 550 nm. Cyclic voltammetry experiments show two ill-defined irreversible oxidation waves around +1.50 and 1.80 V that are assigned to Re(I)/Re(II) and Re(II)/Re(III) couples whereas an irreversible reduction signal centered at -1.80 V is likewise assigned to a ligand reduction process. These results support the proposal of the MLCT nature of the states implied by the emission of the complex. The luminescent decay fits to a biexponential function, where the lifetimes and emission quantum yields are dependent on the solvent polarity. DFT calculations suggest that dπ → π*pyridine and dπ → π*phenyl excited states may account for the existence of two decay lifetimes.

Spectral and kinetic properties of radicals derived from oxidation of quinoxalin-2-one and its methyl derivative.

The kinetics and spectral characteristics of the transients formed in the reactions of •OH and •N3 with quinoxalin-2(1H)-one (Q), its methyl derivative, 3-methylquinoxalin-2(1H)-one (3-MeQ) and pyrazin-2-one (Pyr) were studied by pulse radiolysis in aqueous solutions at pH 7. The transient absorption spectra recorded in the reactions of •OH with Q and 3-MeQ consisted of an absorption band with λmax = 470 nm assigned to the OH-adducts on the benzene ring, and a second band with λmax = 390 nm (for Q) and 370 nm (for 3-MeQ) assigned, inter alia, to the N-centered radicals on a pyrazin-2-one ring. The rate constants of the reactions of •OH with Q and 3-MeQ were found to be in the interval (5.9-9.7) × 109 M-1·s-1 and were assigned to their addition to benzene and pyrazin-2-one rings and H-abstraction from the pyrazin-2-one nitrogen. In turn, the transient absorption spectrum observed in the reaction of •N3 exhibits an absorption band with λmax = 350 nm. This absorption was assigned to the N-centered radical on the Pyr ring formed after deprotonation of the respective radical cation resulting from one-electron oxidation of 3-MeQ. The rate constant of the reaction of •N3 with 3 MeQ was found to be (6.0 ± 0.5) × 109 M-1·s-1. Oxidation of 3-MeQ by •N3 and Pyr by •OH and •N3 confirms earlier spectral assignments. With the rate constant of the •OH radical with Pyr (k = 9.2 ± 0.2) × 109 M-1·s‒1, a primary distribution of the •OH attack was estimated nearly equal between benzene and pyrazin-2-one rings.

Spectral and Kinetic Properties of Radical Cations Derived from Oxoisoaporphines: Relevance to Electron-Transfer Processes Involving Phytoalexins.

The thermally induced intermolecular electron transfer reaction in acetonitrile between the tetracyanoethylene (TCNE), a π-electron acceptor with a large electron affinity, and six oxoisoaporphines (2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one, 5-methoxy-2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one, 1-azabenzo[de]anthracen-7-one, 5-methoxy-1-azabenzo[de]anthracen-7-one, 7H-benzo[e]perimidin-7-one, and 2-methyl-7h-benzo[e]perimidin-7-one) is reported. Spectral and kinetic characteristics are presented for radical cations derived from these six oxoisoaporphines either generated by a thermal reaction or generated radiolytically in argon-saturated 1,2-dichloroethane, oxygen-saturated acetone, and acetonitrile. The radical cations of oxoisoaporphines are insensitive to oxygen and are mostly characterized by absorption maxima of their most intense bands located at λmax = 400-410 nm, except of the radical cations derived from 2,3-dihydrooxoisoaporphines. For the latter compounds, the absorption maxima of the most intense absorption bands are located at λmax = 290-295 nm. Their locations are independent of the presence of functional groups and the solvents used. They are formed in bimolecular processes with pseudo-first-order rate constants ranging from 2.1 × 105 to 1.5 × 106 s-1 (in solutions containing 10-4 M of the substrate), depending on the derivative and the solvent used. They are stable either when formed via the electron-transfer reaction with TCNE or when generated in isolation in pulse radiolysis of Ar-saturated 1,2-dichloroethane. In acetone and acetonitrile they decay predominantly by first-order kinetics with the first-order rate constants ranging from 2.3 × 104 to 5.1 × 104 s-1. Formation of dimeric radical cations for all of the oxoisoaporphines studied was observed in acetonitrile solutions, and for azaoxoisoaporphines also in acetone solutions. The experimental spectra show a reasonably good agreement with the ZINDO/S semiempirical quantum mechanical calculations of radical cation absorptions.

Unexpected imidazoquinoxalinone annulation products in the photoinitiated reaction of substituted-3-methyl-quinoxalin-2-ones with N-phenylglycine.

Photoinduced electron transfer between N-phenylglycine (NPG) and electronically excited triplets of 7-substituted-3-methyl-quinoxalin-2-ones in acetonitrile generate the respective ion radical pair, where by decarboxylation the phenyl-amino-alkyl radical, PhNHCH2•, is generated. This radical reacts with the 3-methyl-quinoxalin-2-ones ground states, leading to the product 2. Other, unexpected, 7-substituted-1,2,3,3a-tetrahydro-3a-methyl-2-phenylimidazo[1,5-a]quinoxalin-4(5H)-ones, annulation products, 3a-f, were generated; likely by the addition of two PhNHCH2• radicals, to positions 3 and 4 of the quinoxalin-2-ones. The reaction mechanism includes a photoinduced one electron transfer initiation step, propagation steps involving radical intermediates and NPG with radical chain termination steps that lead to the respective products 2a-f and 3a-f and NPG by-products. The proposed mechanism accounts for the strong dependency found for the initial photoconsumption quantum yields on the electron-withdrawing power of the substituent. Therefore, photolysis of common reactants widely used such as NPG and substituted quinoxalin-2-ones may provide a simple synthetic way to the unusual, unreported tetrahydro-imidazoquinoxalinones 3a-f.

Photoreduction of azaoxoisoaporphines by amines: laser flash and steady-state photolysis and pulse radiolysis studies.

Photoreduction of 7H-benzo[e]perimidin-7-one (3-AOIA, A1) and its 2-methyl derivative (2-Me-3-AOIA, A2) by non-H-donating amines (1,4-diazabicyclo[2.2.2]octane [DABCO]; 2,2,6,6-tetramethylpiperidine [TMP]), and a hydrogen-donating amine (triethylamine [TEA]), has been studied in deaerated neat acetonitrile solutions using laser flash photolysis (LFP) and steady-state photolysis. The triplet excited states of A1 and A2 were characterized by a strong absorption band with λmax = 440 nm and lifetimes of 20 and 27 µs respectively. In the presence of tertiary amines, both triplet excited states were quenched with rate constants close to the diffusional limit (kq ranged between 10(9) and 10(10) M(-1) s(-1)). The transient absorption spectra observed after quenching with DABCO and TMP were characterized by maxima located at 460 nm and broad shoulders in the range of 500-600 nm. These transient species are attributed to solvent-separated radical ion pairs and/or to isolated radical anions. In the presence of TEA, these transients undergo proton transfer, leading to the neutral hydrogenated radicals, protonated over the N1- and O-atoms. Transient absorption spectra of these transients were characterized by maxima located at 400 and 520 nm and 430 nm respectively. Additional support for these spectral assignments was provided by pulse radiolysis (PR) experiments in acetonitrile and 2-propanol solutions.

Fecal steroid evaluation to monitor reproductive status in wild ungulate females using enzyme immunoassay commercial kits.

Analysis of reproductive hormones in fecal samples is necessary for the noninvasive monitoring of reproductive status in free-ranging species. The aim of the present study was to establish an easy noninvasive method to monitor reproductive status in wild ungulate females. Feces were collected daily, weekly, or three or four times a week directly from the soil for a period ranging from 1 to 9.8 mo. Fecal estradiol and progestagens were monitored in nine wild ungulate females (Barbary sheep, Ammotragus lervia [n = 3]; European bison, Bison bonasus [n = 1]; auroch, Bos taurus primigenius [n = 2]; sitatunga, Tragelaphus spekii gratus [n = 2]; and Indian rhinoceros, Rhinoceros unicornis [n = 1]) by using commercially available enzyme immunoassay kits prepared for human serum or plasma. In the species evaluated in this study, luteal phase, abortion, and gestation patterns corresponded closely with changes in fecal progestagens. Luteal phase and gestation values differed significantly (P < 0.001) from basal values, whereas progestagens values after abortion were not significantly different (P > 0.05) from basal values. For estradiol excretory patterns, follicular phase and pregnancy values differed significantly (P < 0.001) from basal values, but differences between values after abortion and basal values were not significant (P > 0.05); length of estrous cycles were clearly defined through estradiol data. This study demonstrates that ovarian function in the wild ungulate females studied can be monitored by enzyme-linked immunosorbent assay (ELISA). Therefore, ELISA methodologies used here could be a practical alternative to other ELISAs that require more complex procedures or whose commercial availability is difficult.

Photoreduction of oxoisoaporphines by amines: laser flash and steady-state photolysis, pulse radiolysis, and TD-DFT studies.

Photoreduction of oxoisoaporphine (OIA) (1-aza-benzo-[de]anthracen-7-one) and its 5-methoxy (5-MeO-OIA) derivative by selected amines (two non-alpha-hydrogen-donating amines (1,4-diaza[2.2.2]-bicyclooctane (DABCO) and 2,2,6,6-tetramethylpiperidine (TMP)) and three alpha-hydrogen-donating amines (triethylamine (TEA), diethylmethylamine (DEMA), and dimethylethylamine (DMEA))) has been studied in deaerated neat acetonitrile solutions using laser flash and steady-state photolysis. The triplet excited states of OIA and 5-MeO-OIA are characterized by intense absorption maxima located at lambda(max) = 450 nm and lifetimes of 34.7 +/- 0.5 and 44.6 +/- 0.4 micros, respectively. In the presence of tertiary amines, both triplets are quenched with a rate constant that varies from the near diffusion limit (>10(9) M(-1) s(-1)) to a rather low value (approximately 10(7) M(-1) s(-1)) and shows the expected dependence on the reduction potential for one-electron-transfer reactions. The transient absorption spectra observed after quenching of the respective triplet states are characterized by distinct absorption maxima located at lambda(max) = 480 and 490 nm (for OIA and 5-MeO-OIA, respectively) and accompanied by broad shoulders in the range of 510-560 nm. They were assigned to either solvent-separated radical ion pairs and/or isolated radical anions. In the presence of alpha-hydrogen-donating amines these species undergo protonation that leads to the formation of neutral hydrogenated radicals A1H(*)/A2H(*) with two possible sites of protonation, N and O atoms. Pulse radiolysis and molecular modeling together with TD-DFT calculations were used to support the conclusions about the origin of transients.

Transient phenomena in the pulse radiolysis of oxoisoaporphine derivatives in acetonitrile.

The absorption-spectral and kinetic behavior of radical ions and triplet states of two oxoisoaporphine derivatives, 2,3-dihydrooxoisoaporphine (2,3-DHOA) and 5-methoxy-2,3-dihydrooxoisoaporphine (5-MeO-2,3-DHOA), have been studied by UV-vis spectrophotometric pulse radiolysis in a neat acetonitrile saturated with argon and oxygen at room temperature. The radical anions of 2,3-DHOA and 5-MeO-2,3-DHOA are characterized by intense absorption maxima located at lambda max = 605 and 590 nm, with molar absorption coefficients 605 = 5600 M (-1) cm (-1) and 590 = 4900 M (-1) cm (-1), respectively. Both radical anions decay via first-order kinetics with the rate constants in the range (1.5-2.6) x 10 (5) s (-1), predominantly through protonation by adventitious water forming neutral-hydrogenated radicals. Oxygen insensitive, the radical cations of 2,3-DHOA are characterized by a strong nondescript absorption band with no distinct lambda max in the range 350-450 nm. On the other hand, the radical cations of 5-MeO-2,3-DHOA are characterized by the distinctive absorption band with lambda max = 420 nm. The experimental spectra of the neutral-hydrogenated radicals and the triplet excited states derived from 2,3-DHOA and 5-MeO-2,3-DHOA are in accordance with the spectra observed previously during laser flash photolysis ( De la Fuente, J. R. ; et al. J. Phys. Chem. 2005, 109, 5897 ). Most of the transient spectra generated radiolytically are adequately reproduced by quantum mechanical semiempirical PM3 and ZINDO/S methods.

Complete assignments 1H and 13C NMR spectral data of four anabaseine derivatives.

The anabaseine derivatives 6-methoxy-7-hydroxy-1-(pyridin-3-yl)-3,4-dihydroisoquinoline, 6,7-dimethoxy-1-(pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline and 6,7-dimethoxy-1-(piperidin-3-yl)-1,2,3,4-tetrahydroisoquino- line were prepared either by demethylation with HBr or by reduction with different reagents, NaBH4 and H2/PtO2 from 6,7-dimethoxy-1-(pyridin-3-yl)-3,4-dihydroisoquinoline, as starting material. The structures have been fully assigned by the combination of one- and two-dimensional experiments.

Unexpected formation of 1-diethylaminobutadiene in photosensitized oxidation of triethylamine induced by 2,3-dihydro-oxoisoaporphine dyes. A 1H NMR and isotopic exchange study.

[reaction: see text] Photoreduction of oxoisoaporphine dyes occurs via a stepwise mechanism of electron-proton-electron transfer that leads to the N-hydrogen oxoisoaporphine anion. When triethylamine, TEA, was used as the electron donor in anaerobic conditions, 1-diethylaminobutadiene, DEAB, was one of the oxidation products of TEA, among diethylamine and acetaldehyde. DEAB was identified by (1)H NMR and GC-MS experiments by comparison with the authentic 1-diethylaminobutadiene. This is the first report of a butadienyl derivative formed in the dye-sensitized photooxidation of TEA. In addition, isotopic exchange experiments with TEA-d(15) and D(2)O show that the hydrogens at carbon-2 and carbon-4 of the butadienyl moiety are exchangeable. The observed isotopic exchange pattern could be explained by the head-to-tail coupling of an N,N-diethylvinylamine intermediate that exchanges hydrogens at the C-beta via the enammonium ion.

Synthesis and total assignment of 1H and 13C NMR spectra of new oxoisoaporphines by long-range heteronuclear correlations.

The new oxoisoaporphines 7H-dibenzo[de,h]quinolin-7-one, 5-methoxy-7H-dibenzo[de,h]quinolin-7-one, 5-methoxy-6-hydroxy-7H-dibenzo[de,h]quinolin-7-one, 5-hydroxy-7H-dibenzo[de,h]quinolin-7-one and 5-methoxy-6H-dibenzo[de,h]quinolin-6-one were prepared either by oxidation of their 2,3-dihydro derivatives or by heating (2'-(3,4-dihydro-6,7-dimethoxyisoquinolin-1'-yl)phenyl)methylbenzoate with an acetic acid/sulfuric acid mixture at 100 degrees C. The structures were confirmed and 1H and 13C NMR spectra were completely assigned using two-dimensional NMR techniques.

Photoreduction of oxoisoaporphine dyes by amines: transient-absorption and semiempirical quantum-chemical studies.

Photoreduction by amines of oxoisoaporphine dyes occurs via a stepwise mechanism of electron-proton-electron transfer that leads to the metastable N-hydrogen oxoisoaporphine anion. During photoreduction that occurs from the triplet manifold of the oxoisoaporphine, a radical ion A(-)(*), a neutral-hydrogenated radical A-NH(*), and the metastable ion A-NH(-) of the oxoisoaporphine are formed. We present time-resolved spectroscopic data and quantum mechanical semiempirical PM3 and ZINDO/S results for the transient species formed during the flash photolysis of oxoisoaporphines in the presence of amines. These calculations reproduce adequately the experimental spectra of the triplet-triplet absorption near 450 nm, and that of neutral hydrogenated radical of the studied oxoisoaporphines centered at 390 nm. A transient absorption observed near 490 nm, for all of the studied systems, was explained by considering the formation of radical ion pair between the radical anion of the oxoisoaporphine, A(-)(*), and the radical cation of the amine, whose ZINDO/S calculated spectra generate the strongest transition near the experimentally observed absorption maximum at 490 nm, supporting the formation of a radical ion pair complex as the first step of the photoreduction.

Enhancement of riboflavin-mediated photo-oxidation of glucose 6-phosphate dehydrogenase by urocanic acid.

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen-mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.

Photoreduction of oxoisoaporphines. Another example of a formal hydride-transfer mechanism.

Photoreduction of 5,6-dimethoxy-, 5-methoxy- and 2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one (A) by tertiary amines in oxygen-free solutions generates long-lived semi-reduced metastable photoproducts, A-NH(-), via a stepwise electron-proton-electron transfer mechanism with a limit quantum yield of about 0.1 at high TEA concentrations. These metastable photoproducts revert thermally to the initial oxoisoaporphine nearly quantitatively in the presence or absence of oxygen. We present spectrophotometric, NMR and UV-vis data for the metastable photoproducts. The spectrophotometric results and PM3 and ZINDO/S calculations support the proposed mechanism for the photoreduction of the oxoisoaporphines.

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio.

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.

Relative messenger RNA abundance in bovine oocytes collected in vitro or in vivo before and 20 hr after the preovulatory luteinizing hormone surge.

In the cyclic cow, final maturation of the ovulatory follicle is initiated by the preovulatory luteinizing hormone (LH) surge. During the subsequent 24 hr period, the oocyte nucleus undergoes meiotic progression to metaphase II and several changes in cytoplasmic organization take place. We have previously shown that oocytes recovered at the time of the LH peak and matured in vitro are less competent to reach the blastocyst stage than their counterparts recovered 20 hr later following in vivo maturation, despite both groups undergoing IVF and culture in parallel. The objective of this study was to compare, using real-time quantitative RT-PCR, the relative abundance of various developmentally important gene transcripts in these oocytes. The groups used were mature bovine oocytes originating from: (1) 2-6 mm follicles from slaughterhouse ovaries; (2) preovulatory follicles punctured by ovum pick-up just before the LH surge (i.e., immature) and matured in vitro; or (3) preovulatory follicles punctured 20 hr later, just prior to ovulation (i.e., in vivo matured). In addition, immature oocytes from 2-6 mm follicles were examined. We examined the relative mRNA expression of five enzymes involved in protection against free oxygen radicals (mitochondrial Mn-superoxide dismutase, MnSOD, cytosolic Cu/Zn superoxide dismutase, Cu/ZnSOD, gamma-glutamyl-cysteine transferase, GCS, glutathione peroxidase, GPX, sarcosine oxidase, SOX), a transcript involved in follicular development (growth differentiation factor-9, GDF-9), transcripts involved in glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH, glucose transporter type-1 and -8, Glut-1, Glut-8) and genes involved in cell cycle events, Cyclin A and B, and poly(A) polymerase (PAP). Transcripts for all genes were detected, irrespective of oocyte origin. While differences were not significant in all cases, variations in levels of transcript abundance between the groups were related to developmental competence. In particular, transcripts for GDF-9 were expressed at significantly higher levels in oocytes recovered at the LH peak and matured in vitro than in those matured in vivo. The observations with GDF-9 are interesting as this gene is believed to be essential for normal folliculogenesis and may be important in the regulation of early follicle and oocyte growth. In conclusion, the results of this study demonstrate differences in the relative mRNA abundance of several developmentally important gene transcripts in bovine oocytes which may be related to developmental competence.