Enhancing oxygen delivery to ovarian follicles by three different methods markedly improves growth in serum-containing culture medium.

Invitro ovarian follicle culture systems are routinely used to study folliculogenesis and may provide solutions for infertility. Mouse follicles are typically cultured in standard gas-impermeable culture plates under gas phase oxygen concentrations of 5% or 20% (v/v). There is evidence that these conditions may not provide adequate oxygenation for follicles cultured as non-attached intact units in medium supplemented with serum and high levels of FSH. Three different methods of enhancing follicle oxygenation were investigated in this study: increasing the gas phase oxygen concentration, inverting the culture plates and using gas-permeable culture plates. Follicles cultured under 40% O2 were significantly larger (P P P 2 . These effects were associated with reduced secretion of vascular endothelial growth factor (P P P invivo -matured follicles (~500µm in diameter). Such follicular development is not possible under hypoxic conditions.

Mechanism of luminal ATP activated chloride secretion in a polarized epithelium.

There are both secretory and absorptive pathways working in tandem to support ionic movement driving fluid secretion across epithelia. The mechanisms exerting control of fluid secretion in the oviduct is yet to be fully determined. This study explored the role of apical or luminal extracellular ATP (ATPe)-stimulated ion transport in an oviduct epithelium model, using the Ussing chamber short-circuit current (Isc) technique. Basal Isc in oviduct epithelium in response to apical ATPe comprises both chloride secretion and sodium absorption and has distinct temporal phases. A rapid transient peak followed by a sustained small increase above baseline. Both phases of the apical ATPe Isc response are sensitive to anion (HCO3-, Cl-) and cation (Na+) replacement. Additionally, the role of apical chloride channels, basolateral potassium channels and intracellular calcium in supporting the peak Isc current was confirmed. The role of ATP breakdown to adenosine resulting in the activation of P2 receptors was supported by examining the effects of non-hydrolyzable forms of ATP. A P2YR2 potency profile of ATP = UTP > ADP was generated for the apical membrane, suggesting the involvement of the P2YR2 subtype of purinoceptor. A P2X potency profile of ATP = 2MeSATP > alpha,beta-meATP > BzATP was also generated for the apical membrane. In conclusion, these results provide strong evidence that purinergic activation of apical P2YR2 promotes chloride secretion and is thus an important factor in fluid formation by the oviduct.

Intensity matters: effects of cadence and power output on corticospinal excitability during arm cycling are phase and muscle dependent.

The present study investigated the effects of cadence and power output on corticospinal excitability to the biceps (BB) and triceps brachii (TB) during arm cycling. Supraspinal and spinal excitability were assessed using transcranial magnetic stimulation (TMS) of the motor cortex and transmastoid electrical stimulation (TMES) of the corticospinal tract, respectively. Motor-evoked potentials (MEPs) elicited by TMS and cervicomedullary motor-evoked potentials (CMEPs) elicited by TMES were recorded at two positions during arm cycling corresponding to mid-elbow flexion and mid-elbow extension (i.e., 6 and 12 o'clock made relative to a clock face, respectively). Arm cycling was performed at combinations of two cadences (60 and 90 rpm) at three relative power outputs (20, 40, and 60% peak power output). At the 6 o'clock position, BB MEPs increased ~11.5% as cadence increased and up to ~57.2% as power output increased ( P < 0.05). In the TB, MEPs increased ~15.2% with cadence ( P = 0.013) but were not affected by power output, while CMEPs increased with cadence (~16.3%) and power output (up to ~19.1%, P < 0.05). At the 12 o'clock position, BB MEPs increased ~26.8% as cadence increased and up to ~96.1% as power output increased ( P < 0.05), while CMEPs decreased ~29.7% with cadence ( P = 0.013) and did not change with power output ( P = 0.851). In contrast, TB MEPs were not different with cadence or power output, while CMEPs increased ~12.8% with cadence and up to ~23.1% with power output ( P < 0.05). These data suggest that the "type" of intensity differentially modulates supraspinal and spinal excitability in a manner that is phase- and muscle dependent. NEW & NOTEWORTHY There is currently little information available on how changes in locomotor intensity influence excitability within the corticospinal pathway. This study investigated the effects of arm cycling intensity (i.e., alterations in cadence and power output) on corticospinal excitability projecting to the biceps and triceps brachii during arm cycling. We demonstrate that corticospinal excitability is modulated differentially by cadence and power output and that these modulations are dependent on the phase and the muscle examined.

Corrigendum to: Hypoxia limits mouse follicle growth in vitro.

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin-transferrin-selenium (ITS) and I-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P<0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P<0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P>0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL-1 FSH, 25 µgmL-1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 µm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 µm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

Does efavirenz replacement improve neurological function in treated HIV infection?

OBJECTIVES: The contribution of specific antiretroviral drugs to cognitive function in HIV-infected people remains poorly understood. Efavirenz (EFV) may plausibly cause cognitive impairment. The objective of this study was therefore to determine whether chronic EFV therapy is a modifier of neurocognitive and neurometabolic function in the setting of suppressive highly active antiretroviral therapy. METHODS: We performed an open-label phase IV controlled trial. Adult subjects who were stable on suppressive EFV therapy for at least 6 months were switched to ritonavir-boosted lopinavir (LPV/r) with no change in the nucleoside reverse transcriptase inhibitor (NRTI) backbone. The following parameters were assessed before and 10 weeks after therapy switch: cognitive function (by CogState® computerized battery); brain metabolites (by proton magnetic resonance spectroscopy); brain activity [by attentional processing task-based functional magnetic resonance imaging]; and sleep quantity and quality [by sleep diary, Pittsburgh Sleep Quality Index (PSQI) and Epworth Sleepiness Scale]. RESULTS: Sixteen subjects completed the study. Despite most subjects (81%) self-reporting memory problems at baseline, cognitive function, brain metabolites, and brain activity showed no change at 10 weeks after switch. Sleep quality improved on switch off EFV [mean PSQI (standard deviation): EFV, 8.5 (6.5); LPV/r, 5.8 (5.5); mean difference -0.4; 95% confidence interval -6.0 to -0.7]. CONCLUSIONS: This is the first study to assess the effects of chronic EFV therapy on neurological function in a controlled setting. We conclude that EFV withdrawal is unlikely to result in significant modification of neurocognitive function in otherwise stable HIV-infected people.

Hypoxia limits mouse follicle growth in vitro.

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin-transferrin-selenium (ITS) and l-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P < 0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P < 0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P > 0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL-1 FSH, 25 µg mL-1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 µm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 µm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

Results of data gathered at a smoking cessation counselling stand in the Dublin Dental University Hospital on Mouth Cancer Awareness Day 2012.

INTRODUCTION: The addictive aspect of smoking is well acknowledged. Research has shown that interventions by healthcare professionals have been shown to be effective and that smokers will benefit from smoking cessation counselling before, during and after their quit attempts. Dental hygienists, as part of the healthcare team, are well positioned to provide this counselling. MATERIAL AND METHODS: A questionnaire was completed by patients, staff, students and members of the public, during Mouth Cancer Awareness Day 2012 in the Dublin Dental University Hospital to assess the prevalence of smoking as well as the history of smoking and quit attempts by current and former smokers. RESULTS: The prevalence of smoking was lower than the national average. A total of 18.3% of those surveyed were smokers, 25% were former smokers, and 68% of the smokers had their first cigarette within 30 minutes of waking, indicating high dependence. DISCUSSION AND CONCLUSIONS: The majority of the smokers (79%) had attempted to quit. Stress was the most common reason for lapsing. The most common reasons for smoking cessation were health issues. The public is well disposed to receive information regarding smoking and the methods available to quit by healthcare professionals on health awareness days such as Mouth Cancer Awareness Day.

Experimental and theoretical studies of the reaction of the OH radical with alkyl sulfides: 3. Kinetics and mechanism of the OH initiated oxidation of dimethyl, dipropyl, and dibutyl sulfides: reactivity trends in the alkyl sulfides and development of a predictive expression for the reaction of OH with DMS.

A pulsed laser photolysis-pulsed laser-induced fluorescence technique has been employed to measure rate coefficients for the OH-initiated oxidation of dimethyl sulfide (DMS), its deuterated analog (DMS-d(6)), dipropyl sulfide (DPS), and dibutyl sulfide (DBS). Effective rate coefficients have been measured as a function of the partial pressure of O(2) over the temperature range of 240-295 K and at 200 and 600 Torr total pressure. We report the first observations of an O(2) enhancement in the effective rate coefficients for the reactions of OH with DPS and DBS. All observations are consistent with oxidation proceeding via a two-channel oxidation mechanism involving abstraction and addition channels. Structures and thermochemistry of the DPSOH and DBSOH adducts were calculated. Calculated bond strengths of adducts increase with alkyl substitution but are comparable to that of the DMSOH adduct and are consistent with experimental observations. Reactivity trends across the series of alkyl sulfide (C(2)-C(8)) reactions are analyzed. All reactions proceed via a two-channel mechanism involving either an H-atom abstraction or the formation of an OH adduct that can then react with O(2). Measurements presented in this work, in conjunction with previous measurements, have been used to develop a predictive expression for the OH-initiated oxidation of DMS. This expression is based on the elementary rate coefficients in the two-channel mechanism. The expression can calculate the effective rate coefficient for the reaction of OH with DMS over the range of 200-300 K, 0-760 Torr, and 0-100% partial pressure of O(2). This expression expands on previously published work but is applicable to DMS oxidation throughout the troposphere.

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model.

Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal's diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of Δ-9 desaturase in vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-γ, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, Δ-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 µM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration (P < 0.01). Expression of Δ-9 desaturase mRNA was decreased five- and seven-fold, respectively, following 50 and 100 µM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6- and 18-fold in cells supplemented with 50 and 100 µM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both Δ-9 desaturase (P < 0.001) and SREBP-1c (P < 0.001), while a significant positive relationship was observed between Δ-9 desaturase and SREBP-1c gene expression (P < 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both Δ-9 desaturase and SREBP-1c in vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

Experimental and theoretical studies of the reaction of the OH radical with alkyl sulfides: 2. Kinetics and mechanism of the OH initiated oxidation of methylethyl and diethyl sulfides; observations of a two channel oxidation mechanism.

A pulsed laser photolysis-pulsed laser induced fluorescence technique has been employed to measure rate coefficients for the OH initiated oxidation of methylethyl sulfide (MES) and diethylsulfide (DES). In the absence of oxygen and at low sulfide concentrations we measure rate coefficients that are independent of pressure and temperature. At high sulfide concentrations and a temperature of 245 K, we observed the equilibration of MESOH and DESOH adducts over the pressure range 100-600 Torr. In the presence of O(2) the observed rate coefficients show a dependence on the O(2) partial pressure. We measured the dependence of the overall rates of oxidation on the partial pressure of O(2) over the temperature range 240-295 K and at 200 and 600 Torr total pressures. All observations are consistent with oxidation proceeding via a two channel oxidation mechanism involving abstraction and addition channels, analogous to that observed in the OH initiated oxidation of dimethylsulfide (DMS). Structures and thermochemistry of the MESOH and DESOH adducts were calculated and all results compared to those for DMS. Calculated bond strengths of adducts increase with alkyl substitution but are comparable to that of the DMSOH adduct and are consistent with experimental observations.

Experimental and theoretical studies of the reaction of the OH radical with alkyl sulfides: 1. Direct observations of the formation of the OH-DMS adduct-pressure dependence of the forward rate of addition and development of a predictive expression at low temperature.

A pulsed laser photolysis-pulsed laser-induced fluorescence (PLP-PLIF) system was employed to study the kinetics and mechanisms of reactions (1) OH + h6-DMS --> products and (2) OH + d6-DMS --> products. We report direct observations of the rate coefficients for the formation and dissociation of the h6-OHDMS and d6-OHDMS adducts over the pressure range 50-650 Torr and between 240 and 245 K, together with measurements of the oxygen dependence of the effective rate coefficients for reactions 1 and 2 under similar conditions. The effective rate coefficients increased as a function of O2 concentration reaching their limiting values in each case. The values of the adduct formation rate, obtained from the O2 dependencies, were in excellent agreement with values determined from direct observation of adduct equilibration in N2. OH regeneration is insignificant. The rate coefficients for the formation of the adduct isotopomers showed slight differences in their falloff behavior and do not approach the high-pressure limit in either case. The equilibrium constants obtained show no dependence on isotopomer and are in good agreement with previous work. A "second-law" analysis of the temperature dependence of the equilibrium constant gives an adduct bond strength (DeltaH degrees =-10.9 +/- 1.0 kcal mol(-1)), also in good agreement with previously reported values. Using the entropy calculated from the ab initio vibrational frequencies, we obtain a "third-law" value for the reaction enthalpy at 240 K, DeltaH(240K) degrees = -10.5 kcal mol(-1) in good agreement with the other approach. The rate coefficient for the reactions of the adducts with O2 was obtained from an analysis of the O2 dependence and was determined to be 6.3 +/- 1.2 x 10(-13) cm3 molecule(-1) s(-1), with no dependence on pressure or isotopomer. The pressure and temperature dependence for all of the elementary processes in the initial steps of the dimethylsulfide (DMS) oxidation mechanism have been characterized in the range 238-245 K, allowing the formulation of an expression which can be used to calculate the effective rate coefficient for reaction 1 at any pressure and oxygen concentration. The expression can calculate the effective rate coefficient for reaction 1 to +/- 40% over the range 220-260 K, with the largest errors at the extremes of this range. Gaussian 03 has been used to calculate the structure of the OH-DMS adduct and its deuterated isotopomer. We find similar bound structures for both isotopomers. The calculated enthalpies of formation of the adducts are lower than the experimentally determined values.

Increased local matrix metalloproteinase-8 expression in the periodontal connective tissues of smokers with periodontal disease.

Matrix metalloproteinase (MMP)-8 has been associated with the progression of periodontitis, a common inflammatory disease of the supporting structures of the teeth, and with other degradative diseases. Tobacco smokers are at high risk of developing periodontitis that may progress more rapidly and respond poorly to treatment. Therefore, MMP-8 expression was determined by immunofluorescence staining in 60 random, computer-selected fields in the excised periodontal tissues of smokers and non-smokers, balanced for age, gender, and periodontal status. Immunofluorescence intensity, representing MMP-8 expression, in the periodontal tissues of smokers (30 fields from 6 subjects, mean 1154+/-124 units) was significantly higher than that in the periodontal tissues of non-smokers (30 fields from 6 subjects, mean 817+/-60 units; p < 0.05). Serum MMP-8 concentrations were measured by ELISA and compared in a larger group of smokers (n = 20) and age- and gender-balanced non-smokers (n = 20). Systemic MMP-8 concentrations in smokers and non-smokers were not significantly different (p > 0.05). A local tobacco-related increase in MMP-8 burden may contribute to periodontal disease progression in tobacco smokers. This finding may also have relevance to other tobacco-induced inflammatory diseases, such as vascular and pulmonary diseases.

Oxidative phosphorylation and the tricarboxylic acid cycle are essential for normal development of mouse ovarian follicles.

BACKGROUND: Mouse ovarian follicles are typically grown in upright drops of culture medium. Recently we found that culture of follicles at the medium-gas interface in inverted drops markedly improved follicular development, possibly due to improved access of oxygen to the follicle. In this study, we examined the importance of aerobic energy metabolism for follicle development by culturing mouse follicles (198 6 16.5 initial microm diameter, mean 6 SD) in the presence of phosphorylation and tricarboxylic acid (TCA) cycle inhibitors. METHODS: All inhibitors were tested in the inverted system using 100 microl medium drops in 96-well plates; certain inhibitors were also tested in upright drops with or without an oil overlay. RESULTS: The oxidative phosphorylation inhibitor rotenone (0.1, 0.5 and 1 micromol/l) totally abolished follicle growth in the inverted system; cyanide (1 mmol/l) totally abolished growth in the upright with oil system but not in the inverted system (possibly due to loss of cyanide gas due to the absence of an oil overlay). The mitochondrial uncoupler 2,4-dinitrophenol (0.5 and 1 mmol/l) also abolished growth in the inverted system. The TCA cycle inhibitor monofluoroacetate (10 mmol/l), significantly inhibited growth in all three culture systems (P < 0.01) but malonate (10 mmol/l) had no effect. CONCLUSIONS: Aerobic metabolism and an adequate oxygen supply are essential for normal follicular development.

A novel follicle culture system markedly increases follicle volume, cell number and oestradiol secretion.

This study reports a novel, simple method for culture of mouse follicles which results in follicles with cell numbers similar to in vivo fully grown follicles. Using this method, follicles (180-240 microm in diameter) were cultured in a 100 microl inverted drop of medium without oil and compared with culture in upright drops with and without a mineral oil overlay. Follicles, isolated from C57BL/6 x CBA/ca crossbred and MF1 inbred mice, were cultured individually at 37 degrees C in 96-well round-bottomed suspension cell tissue culture plates for 6 days. Follicles grown in the inverted drop culture system reached a markedly higher final diameter (means+/-s.e.m.; 471 +/- 6.0 microm) as compared with the upright with oil (363 +/- 2.7 microm) and without oil (358 +/- 4.0) systems. There was no significant effect of mouse strain on follicle diameter. Follicular secretion of oestradiol and lactate into the medium was measured on days 2, 4 and 6 of culture. Secretion of oestradiol per follicle on day 6 was 2.49 +/- 0.45 ng in the inverted and 0.90 +/- 0.17 ng in the upright without oil system (P < 0.001). Follicular secretion of lactate on a per unit of follicle volume basis remained constant in the inverted system over days 2, 4 and 6 and was less (P < 0.001) than secretion in both the upright with and without oil systems. Follicle cell proliferation was markedly increased in the inverted as compared with the upright with oil system; the increases in cell numbers were significant on day 3 (P < 0.01) and on all subsequent days (P < 0.001). These results are discussed in relation to the supply of oxygen to the follicle in culture.

Rapid, ultra-sensitive detection of gas phase elemental mercury under atmospheric conditions using sequential two-photon laser induced fluorescence.

We have examined the sensitivity of sequential two photon laser induced fluorescence (LIF) detection of elemental mercury, Hg(0) in the gas phase. The most sensitive approach involves an initial laser excitation of the 6(3)P1-6(1)S0 transition at 253.7 nm, followed by excitation with a second laser to the 7(1)S0 level. Blue shifted fluorescence is observed on the 6(1)P1-6(1)S0 transition at 184.9 nm. The excitation scheme, involving sequential excitation of two atomic transitions, followed by detection of the emission from a third is extremely specific and precludes detection of anything other than atomic mercury. Using our 10 Hz laser system we have achieved a detection sensitivity of 0.1 ng m(-3) at a sampling rate of 0.1 Hz, i.e. averaging 100 laser shots at a pressure of one atmosphere in air. At low concentrations we sampled simultaneously with an automated mercury analyzer (Tekran 2537A), to ensure accuracy. We have examined the linearity of the technique, generating flows containing mercury concentrations between 1 and 10,000 ng m(-3) using a permeation tube and dynamic dilution, but relying on the concentrations given by the Tekran at low levels and the concentration calculated from dilution at high levels. We find that the detection is linear over the five orders of magnitude that we were able to vary the concentration. Our measured detection limits in He and Ar are much lower as these gases are inefficient fluorescence quenchers.

The phosphatidylinositol signalling system in elongating bovine blastocysts; formation of phosphoinositides, inositol phosphates and stimulation by growth factors.

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo elongating cattle blastocysts was investigated using [3H]myo-inositol. Uptake was examined in 13-, 14- and 16-day-old blastocysts and was largely sodium-dependent throughout (P<0.001), indicating the presence of a sodium-dependent inositol transporter. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP and PtdInsP2, and the inositol phosphates of the phosphatidylinositol signal transduction system was examined at Days 14 and 16; incorporation into the three phosphoinositides and into the inositol phosphate species, InsP1, InsP2, InsP3 (including the second messenger, Ins(1,4,5)P3) and InsP4 was detected in both blastocyst stages. The effects of the peptide growth factor, epidermal growth factor (EGF), and the lipid growth factors, lysophosphatidic acid (LPA) and platelet activating factor (PAF), on the activity of the phosphatidylinositol signalling system in 14- and 16-day-old blastocysts were examined. All growth factors significantly stimulated phosphatidylinositol signalling activity. Epidermal growth factor was stimulatory (P<0.001) only in 16-day-old blastocysts, whereas LPA and PAF were active in both 14- (P<0.005 for LPA and P<0.001 for PAF) and 16-day-old blastocysts (P<0.001 for LPA and PAF). These results indicate that the phosphatidylinositol signalling system is present in cattle blastocysts at the elongation stage and is responsive to stimulation by growth factors.

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages.

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.

Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.