The effects of EGF and IGF-1 on FSH-mediated in vitro maturation of domestic cat oocytes derived from follicular and luteal stages

The objective of this study was to evaluate the influence of epidermal growth factor (EGF) and insulin like growth factor-I (IGF-1) on the in vitro maturation of cat oocytes recovered from follicular and luteal stage ovaries. Oocytes from follicular (n = 580) and luteal (n = 209) stages were harvested and divided into four groups, which were cultured in FSH-mediated maturation medium supplemented with: (1) EGF alone (25 ng/mL); (2) IGF-1 alone (100 ng/mL); (3) EGF + IGF-1 (25 ng/mL EGF + 100 ng/mL IGF-I); or (4) no growth factor (control). The proportion of follicular stage oocytes reaching the metaphase II stage was significantly higher than that of oocytes obtained at the luteal stage in both control and study groups (p < 0.001). The percentages of oocytes reaching the metaphase II stage during the follicular period were 62.6% in control; 70.9% in EGF; 72.8% in IGF-1, and 78.1% in EGF + IGF-1 groups, whereas the respective values for gametes collected from luteal stage ovaries were 12.5%, 17.5%, 12.5%, and 16.9%. Additionally, the differences between the study and control groups were significant in the case of follicular stage oocytes. Finally, supplementing the maturation medium with EGF and/or IGF-1 significantly enhanced the meiotic maturation of oocytes recovered from follicular stage ovaries. The present study also demonstrated that the combination of EGF and IGF-I provides an additional or synergic effect on meiotic maturation of oocytes recovered from the follicular stage.     

In vitro survival and development of goat preantral follicles in two different oxygen tensions

The aim of the present study was to evaluate the effect of two different oxygen (O₂) concentrations on survival and development of preantral follicles of goats cultured in vitro. Preantral ovarian follicles (≥150 μm) were isolated from ovarian cortex fragments of goats and individually cultured for 30 days under two different O₂ concentrations (5% and 20% O₂). Follicle development was evaluated on the basis of antral cavity formation, increase in follicular diameter, presence of healthy cumulus oocyte complexes and fully grown oocytes. Results showed with progression of culture period from 6 to 12 days, a decrease in follicular survival was observed in both O₂ concentrations (P < 0.05). When the O₂ tensions were compared to each other in the different days of culture, 20% O₂ was more efficient in promoting an increase in follicular diameter from day 24 of culture onward than 5% O₂ (P < 0.05). However, follicles cultured with 5% O₂ had an increased percentage of antrum formation from 12 days to the end of culture, compared with 20% O₂ (P < 0.05). Moreover, there was no difference in percentage of fully developed oocytes with the different O₂ tensions. However, only oocytes (16.7%) from follicles cultured in 20% O₂ resumed meiosis. In conclusion, concentration of 20% O₂ was more efficient in promoting follicular growth and oocyte meiosis resumption from preantral follicles of goats when grown in vitro.     

Effects of testosterone and 2-hydroxyflutamide on progesterone receptor expression in porcine ovarian follicles in vitro

The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T + 2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6–8 mm in diameter) were cultured for 48 h. Experimental cultures were carried out with the addition of T (10^?7 M), 2-Hf (1.7 × 10^?4 M) or both T and 2-Hf for the last 24 h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6–8 mm in diameter) were cultured for 24 h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P₄) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P₄ secretion, but simultaneous addition of 2-Hf and T increased P₄ secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.     

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 °C while the other was transported at 37 °C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto–orcein staining.Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI + MII) than the oocytes from anestrual ovaries in the 37 °C group (p < 0.05). However, oocytes harvested from anestrual ovaries transported at 4 °C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant (p < 0.05). The oocytes from anestrual ovaries transported at 4 °C have significantly higher maturation rates than those transported at 37 °C (p < 0.0001). However, the transport temperature (37 or 4 °C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal (p = 0.61) and follicular (p = 0.48) stage ovaries.It can be concluded from this study that (1) both transport temperature and transport temperature × estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 °C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.     

Magnesium uptake of Arabidopsis transporters,AtMRS2-10 and AtMRS2-11, expressed in Escherichia coli mutants: Complementation and growth inhibition by aluminum

Magnesium (Mg^2 +) plays a critical role in many physiological processes. Mg^2 + transport systems in Salmonella have been well documented, but those in Escherichia coli have not been fully elucidated. We examined the effects of corA, mgtA, yhiD and corC gene deletion on Mg^2 + transport in E. coli. We obtained every combination of double, triple and quadruple mutants. The corA and mgtA double mutant required addition of 10 mM Mg^2 + to Luria-Bertani (LB) medium for growth, and the corA, mgtA and yhiD triple mutant TM2 required a higher Mg^2 + concentration. The Mg^2 + requirement of the quadruple mutant was similar to that of TM2. The results demonstrated that either CorA or MgtA is necessary for normal E. coli growth in LB medium and that YhiD plays a role in Mg^2 + transport under high Mg^2 + growth conditions in E. coli. The Arabidopsis Mg^2 + transporters, AtMRS2-10 and AtMRS2-11, were heterologously expressed in TM2 cells. TM2 cells expressing AtMRS2-10 and AtMRS2-11 could grow in LB medium that had been supplemented with 1 mM Mg^2 + and without Mg^2 + supplementation, respectively, and cell growth was inhibited by 2 mM AlCl₃. The results indicated that the growth of TM2 expressing AtMRS2-10 and AtMRS2-11 reflected these AtMRS2 function for Mg^2 + and aluminum. The E. coli TM2 cells are useful for functional analysis of Arabidopsis MRS2 proteins.     

Inventory and input–output analysis of CO2 emissions by fossil fuel consumption in Beijing 2007

With the most recent statistics available, a concrete emissions inventory is compiled for an input–output analysis to investigate the embodied CO₂ emissions induced by fossil fuel combustion of Beijing economy in 2007. Results show that the total direct CO₂ emissions amount to 9.45E + 07 t, within which 56.81% are released from coal combustion, 11.50% from coke combustion, 9.03% from kerosene combustion, 8.70% from natural gas and 6.40% from diesel, respectively. The average intensity of secondary industries (3.12 t/1E + 4 Yuan) is 0.65 times larger than that of primary industries (1.89 t/1E + 4 Yuan) and 1.58 times larger than that of tertiary industries (1.21 t/1E + 4 Yuan). The sector of Construction Industry contributes the largest share (21.98%) of CO₂ emissions embodied in final demand for Beijing due to its considerable capital investment. Beijing is a net importer of embodied CO₂ emissions with total import and export of 3.06E + 08 and 2.00E + 08 t, respectively. Results of this study provide a sound scientific database for effective policy making in Beijing to reduce CO₂ emissions.     

Embryo production in superstimulated llamas pre-treated to inhibit follicular growth

Llamas are monotocous and the length of their gestation period varies between 342 and 350 days. Thus the average number of offspring any female can produce throughout her reproductive life is very limited to spread a desired genome. The multiple ovulation and embryo transfer (MOET) technique allows an alternative to this limitation and reduces the generation interval. The objective of this study was to evaluate embryo recovery in superstimulated llamas which had previously been hormone-treated to inhibit follicular growth. A total of 50 female llamas were monitored daily via rectal palpation and ultrasound and divided according to their ovarian follicular growth into four phases. The females in each phase were then randomly divided into two groups: A (n = 20) received a single dose of 1 mg of estradiol benzoate (EB) on the first day of the treatment + 100 mg of progesterone (P4) i.m. for 5 days with 5 animals per phase and B (n = 20) received 1 mg EB at onset + 150 mg P4 i.m. for a period of 5 days with 5 animals per phase. Group C (n = 10) or control did not receive any prior hormonal treatment and the females were in follicular phase I. All groups were monitored daily and, in the presence of ovarian follicles smaller than the dominant size at the end of treatment, all were superstimulated with 1000 IU eCG. For plasma progesterone concentration recording, daily blood samples were collected from days ?1 to 5 in the treated females in Group A and B. No significant differences were observed regarding the inhibition of follicle growth and in the plasma progesterone concentrations between Group A and B. The ovarian response to superstimulation was 56.2%, 71.4% and 90%, with the average number of dominant follicles produced per female being 4.4 ± 0.9; 4.8 ± 0.7 and 4.6 ± 0.6 in Groups A, B and C, respectively. The embryo recovery rate was 77.7%; 90% and 66.7% and the average number of embryos recovered per female was 2.9 ± 0.9; 2.6 ± 0.9 and 2.4 ± 0.8 for Groups A, B and C, respectively. In Groups A and B, the static follicular phase (III) seemed to be ideal for initiating the assisted reproductive technique of MOET. Although prior administration of P4 + EB seems to have no effect on the number of females that responded to the superstimulation treatments, the number of embryos recovered showed a tendency to be higher when ovarian follicle growth inhibition was performed beforehand.     

Breast cancer aromatase expression evaluated by the novel antibody 677: Correlations to intra-tumor estrogen levels and hormone receptor status

Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E₂; estrone, E₁; estrone sulfate, E₁S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E₂ levels comparing 677+ versus 677? tumors, although a non-significant trend towards higher tumor E₁ and E₁S levels was observed in 677+ breast cancers. In contrast, tumor levels of E₂ were significantly higher in ER+ tumors compared to ER? tumors (P < 0.001) and to benign breast tissue from the same breast (P < 0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E₂ (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E₁ (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E₁S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677? tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E₂ were also found to be significantly higher among 677+ compared to 677? tumors: 873.2 fmol/g (95% CI 395.9–1925.6) versus 217.9 fmol/g (95% CI 88.8–534.9) (P = 0.015).In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.     

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO₂ in air for 40–44 h. No significant difference (P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased (P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced (P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser (P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced (P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.     

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD_25–56 and preproIMD_57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin_45–92 (MRproAM_45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml⁻¹) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml⁻¹; r = 0.49, p < 0.001). Plasma preproIMD_25–56 (39.6 + 3.1 pg ml⁻¹), preproIMD_57–92 (25.9 + 3.8 pg ml⁻¹) and MRproAM_45–92 (200.2 + 6.7 pg ml⁻¹) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml⁻¹, p < 0.05), similarly to MRproAM_45–92 (329.5 + 41.9 pg ml⁻¹, p < 0.001) and AM (56.8 + 10.9 pg ml⁻¹, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml⁻¹) than those without (6.5 + 1.0 pg ml⁻¹; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.     

Influence of oocyte donor on in vitro embryo production in buffalo

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n = 40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A + B COC recorded in each 4-PS period had great repeatability (r = 0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A + B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r = 0.60; P < 0.001), SFL (r = 0.68; P < 0.001), COC (r = 0.48; P < 0.01) and Grade A + B COC (r = 0.40; P < 0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P < 0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r = 0.80; P < 0.05) and COC (r = 0.76; P < 0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.     

Comparison of the patterns of antral follicular development between hormonally synchronized and natural estrous cycles of non-seasonal,polyestrous goats in the tropics

The effects of estrus synchronization with prostaglandin F_2α (PGF_2α) and Controlled Internal Drug Release Device (CIDR) on ensuing antral follicular development were documented and compared to natural estrous cycles of non-seasonal tropical goats. Two to six follicular waves were observed, with the three-follicular wave pattern being most frequently observed (58%), followed by four follicular waves (31.6%) per estrous cycle. There were no significant differences (p > 0.05) between the PGF_2α- or CIDR-synchronized and natural estrous cycles nor between the synchronized and subsequent non-synchronized cycles in terms of the time of ovulation, the duration of inter-ovulatory intervals, daily numbers of antral follicles ≥3 mm in diameter, and the number of follicular waves per cycle in the goats of the present study.     

Temporal and spatial variability in nitrogen uptake kinetics during harmful dinoflagellate blooms in the East China Sea

Temporal and spatial variability in the kinetic parameters of uptake of nitrate (NO₃⁻), ammonium (NH₄⁺), urea, and glycine was measured during dinoflagellate blooms in Changjiang River estuary and East China Sea coast, 2005. Karenia mikimotoi was the dominant species in the early stage of the blooms and was succeeded by Prorocentrum donghaiense. The uptake of nitrogen (N) was determined using ¹⁵N tracer techniques. The results of comparison kinetic parameters with ambient nutrients confirmed that different N forms were preferentially taken up during different stages of the bloom. NO₃⁻ (V_max 0.044 h⁻¹; K_s 60.8 μM-N) was an important N source before it was depleted. NH₄⁺ (V_max 0.049 h⁻¹; K_s 2.15 μM-N) was generally the preferred N. Between the 2 organic N sources, urea was more preferred when K. mikimotoi dominated the bloom (V_max 0.020 h⁻¹; K_s 1.35 μM-N) and glycine, considered as a dominant amino acid, was more preferred when P. donghaiense dominated the bloom (V_max 0.025 h⁻¹; K_s 1.76 μM-N). The change of N uptake preference by the bloom-forming algae was also related to the variation in ambient N concentrations.     

Effect of FSH on E2/GPR30-mediated mouse oocyte maturation in vitro

Mammalian oocyte restores meiosis can be stimulated by follicle-stimulating hormone (FSH) under normal physiological conditions. G-protein coupled receptor 30 (GPR30), an non-classical estrogen membrane receptor, has been widely reported in teleost oocyte maturation. However, it remains unknown whether GPR30 involves the role of FSH in mammalian cumulus expansion and oocyte maturation. Here, we used mouse cumulus-oocyte complexes (COCs) as a model to investigate how FSH affects the in vitro maturation of mouse oocytes mediated by 17β-estradiol (E₂)/GPR30 signaling. Our study reveals that FSH starts regulating mouse cumulus expansion precisely at 8 h in in vitro culture. ELISA measurement of E₂ levels in culture medium revealed that FSH activated aromatase to promote E₂ production in vitro in cultured mouse COCs. Moreover, the results of real-time quantitative PCR indicated that FSH-induced in vitro maturation of mouse oocytes was regulated by the estrogen-signaling pathway mediated by GPR30; FSH treatment markedly increased the mRNA expression of HAS2, PTGS2, and GREM1 in COCs. Exploration of the underlying mechanism suggested that E₂ produced by mouse COCs regulated the phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2) through GPR30 and thereby promoted mouse cumulus-cell expansion and oocyte maturation. In conclusion, our study reveals that FSH induced estrogen production in mouse COCs through aromatase, and that aromatase/GPR30/ERK1/2 signaling is involved in FSH-induced cumulus expansion.     

Expression of receptors for BMP15 is differentially regulated in dominant and subordinate follicles during follicle deviation in cattle

Bone morphogenetic proteins are known to be involved in determining ovulation rate in mammals. The mechanisms through which these proteins determine follicle fate are incompletely understood. In the present study, we used cattle as a model to evaluate the regulation of BMP15 and GDF9 receptors in granulosa cells during dominant follicle (DF) selection. Before follicular deviation (day 2 of the follicular wave), BMPR2 mRNA abundance tended to be higher in the second largest follicles (F2; P < 0.1) compared to the future dominant follicle (F1). At the expected time of follicular deviation (day 3), BMPR2 and BMPR1B mRNA levels were higher in subordinate follicles (SFs; P < 0.05) compared to dominant follicles (DFs). After deviation (on day 4), BMPR1B mRNA and protein were significantly more abundant in atretic SFs (as assessed by cleaved caspase 3) than in DFs. The fact that BMPR1B is more expressed in atretic follicles was further confirmed by using intrafollicular treatment with two agents known to induce atresia, namely an estradiol receptor antagonist (fulvestrant) and FGF10. In conclusion, the fact that BMPR-1B and -2 are more expressed in the second largest follicles before and at the expected time of follicular deviation is indicative of their inhibitory role in follicle differentiation and steroidogenesis. BMPR1B also seems to have a pivotal role during follicle regression since it is upregulated in advanced atretic follicles.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

The combination of basic fibroblast growth factor and kit ligand promotes the proliferation,activity and steroidogenesis of granulosa cells during human ovarian cortical culture

Different factors, such as basic fibroblast growth factor (bFGF) and kit ligand (KL), are used in ovarian cortical culture to promote activation of primordial follicles. In the present study, the effects of bFGF and KL, alone and in combination, were evaluated on human follicular activation and growth during in-situ cortical culture. Slow frozen-thawed human ovarian cortical tissues (n = 6) were cultured in 4 different groups: 1) control (base medium), 2) KL (base medium; BM + 100 ng/ml KL), 3) bFGF (BM + 100 ng/ml bFGF) and 4) bFGF + KL (BM + 100 ng/ml KL + 100 ng/ml bFGF) for a week. The proportion of morphologically normal and degenerated follicles at different developmental stages, secreted hormonal levels and specific gene expressions were compared. Although the proportion of growing follicles was higher than primordial counterpart in all cultured groups, no significant differences were observed among the cultured groups. In all cultured groups, anti-Müllerian hormone (AMH), progesterone and estradiol hormones levels increased after 7 days of culture; however, this increase was only significant for estradiol in the bFGF + KL group. The expression of Ki67 gene indicated an increase in ovarian cell proliferation in the three experimental groups compared to the control group, however this increment was only significant for the bFGF + KL group. It can be concluded that KL and bFGF factors individually have no beneficial effects on in-situ follicular growth, but their combination positively influences steroidogenesis of granulosa cells without significantly increasing the number of growing follicles.     

Large conductance Ca2+-activated K+ channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects

Large conductance calcium activated potassium channels (BK_Ca) are fundamental in the control of cellular excitability. Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BK_Ca channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BK_Ca channel α-subunit alone or α + β1-subunit complex.Channel activity was determined using a non-radioactive Rb⁺ efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb⁺ efflux both in cells expressing α-subunit alone or α + β1-subunits. Co-expression of the β1-subunit modified the Rb⁺ efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC₄₀ values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BK_Ca channel α + β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC₄₀ values when tested on the BK_Ca α + β1-subunit expressing cells compared to BK_Ca α-subunit expressing cells. Further, the E_max values for BKPr4, BKVV and BKH1 were lower in the BK_Ca channel α + β1-subunit expressing cells.In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BK_Ca channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.     

Synthesis,spectroscopic and redox properties of some ruthenium(II) thiosemicarbazone complexes: Structural description of four of these complexes

Sixteen neutral mixed ligand thiosemicarbazone complexes of ruthenium having general formula [Ru(PPh₃)₂L₂], where LH = 1-(arylidine)4-aryl thiosemicarbazones, have been synthesized and characterized. All complexes are diamagnetic and hence ruthenium is in the +2 oxidation state (low-spin d⁶, S = 0). The complexes show several intense peaks in the visible region due to allowed metal to ligand charge transfer transitions. The structures of four of the complexes have been determined by single-crystal X-ray diffraction and they show that thiosemicarbazone ligands coordinate to the ruthenium center through the hydrazinic nitrogen and sulfur forming four-membered chelate rings with ruthenium in N₂S₂P₂ coordination environment. In dichloromethane solution, the complexes show two quasi-reversible oxidative responses corresponding to loss of electron from HOMO and HOMO − 1. The E⁰ values of the above two oxidations shows good linear relationship with Hammett substituents constant (σ) as well as with the HOMO energy of the molecules calculated by the EHMO method. A DFT calculation on one representative complex suggests that there is appreciable contribution of the sulfur p-orbitals to the HOMO and HOMO − 1. Thus, assignment of the oxidation state of the metal in such complexes must be made with caution.