Cryopreservation of human ovarian tissue: Comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Validation of a quantitative assay for human neutrophil peptide-1, -2, and -3 in human plasma and serum by liquid chromatography coupled to tandem mass spectrometry

A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography–tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 μl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C₁₈ column (50 mm × 2.1 mm I.D., particle size 3.5 μm), using a water–methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.     

Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoidea on human ovarian cancer cell lines

Margaritaria discoidea (Baill.) G. L. Webster (Euphorbiaceae) is a well-known medicinal plant in Africa used for the treatment of various diseases. So far, no cytotoxic effects of plant extracts on cancer cell lines have been reported.Aim of the studyTo evaluate the cytotoxicity against human ovarian cancer cells of extracts of M. discoidea and characterize the major bioactive compounds.MethodsBoth organic and aqueous extracts of this plant were obtained by maceration. The sulforhodamine B cell proliferation assay was used for evaluation of their cytotoxic activities and the potential bioactive compounds were characterized by gas chromatography–mass spectrometry.ResultsThe organic extract of M. discoidea showed stronger cytotoxicity than the aqueous extract with IC₅₀ values of 14.4 ± 3.0, 14.2 ± 1.2 and 34.7 ± 0.5 µg/ml on OVCAR-8, A2780 and cisplatin-resistant A2780cis ovarian cancer cells, respectively. The organic extract was further subjected to bioassay-guided fractionation by partitioning with n-hexane, ethyl acetate, and n-butanol in water. The ethyl acetate fraction was the most potent on the three ovarian cancer cell lines. A GC–MS analysis of trimethylsilyl derivatives of this fraction indicated the presence of phenolic compounds such as gallic acid and the alkaloid securinine. The IC₅₀ values of these two compounds were determined to be in the range of 3–16 µM, which indicated that they could contribute to the cytotoxic activity of the extract of M. discoidea.ConclusionsThis study has evaluated the cytotoxicity of stem bark extracts of M. discoidea against ovarian cancer cells and provided a basis of further development of this plant for the treatment of ovarian cancer.     

Toxic and genotoxic effects of potassium dichromate in Pseudokirchneriella subcapitata detected by microscopy and AFLP marker analysis

The effect of chromium on Pseudokirchneriella subcapitata has been assessed using different approaches: growth rate, metabolic activity microscopy analyses, and amplified fragment length polymorphism (AFLP) assessment of DNA damage. Starting from 24 h of treatment all the tested concentrations resulted in consistent algal growth inhibition. The average daily growth rate after 72 h calculated for the control (0.53 ± 0.01) was statistically higher than those estimated for cell treated with 1, 2.5, 5 and 7.5 μg g⁻¹ potassium dichromate (0.46 ± 0.02; 0.32 ± 0.01; 0.25 ± 0.01 and −0.02 ± 0.04, respectively). A reduction of viable cell numbers, estimated with FDA approach, was also observed after 24 h treatment for all the tested chromium concentrations apart from the lowest (1 μg g⁻¹ potassium dichromate). A recovery of esterase activity was detected after 48 and 72 h for all treatments with the except of the 7.5 μg g⁻¹ potassium dichromate treated samples showing a very modest recovery. This data suggests that potassium dichromate is, even from the lowest tested concentration, highly toxic to P. subcapitata, and that this algal strain is a sensitive organism suitable for monitoring chromium in water. Our data also suggest that although algal counting by microscope and the FDA test gave similar indications concerning chromium effect, the FDA test was not completely reliable. In fact, we observed a decrease in the FDA stained cell numbers in control samples after 48 and 72 h of treatment, when most cells were actively proliferating. This lack of fluorescence could be explained by an uncontrolled fluorescein efflux due to the interaction of many experimental factors. The genotoxic effects of the different chromium concentrations were investigated by analyses of the DNA from control and treated algal samples, 72 h after inoculation. AFLP analysis revealed a total of 258 bands, 109 of which were polymorphics. Analysis of the AFLP matrix suggests that potassium dichromate is a powerful genotoxic agent, inducing genetic mutations also at the lowest tested concentration (0.35 μg g⁻¹). Furthermore, we observed a correlation between the polymorphic bands with increasing chromium concentration. Finally, the absence of preferential mutation sites suggests that the chromium induced DNA changes are randomly distributed in the genome.     

Genetic polymorphisms in AURKA,BRCA1, CCNE1 and CDK2 are associated with ovarian cancer susceptibility among Chinese Han women

IntroductionCentrosome aberrations and cell-cycle deregulation have important implications for ovarian cancer development. The AURKA, BRCA1, CCNE1 and CDK2 genes play pivotal roles in centrosome duplication and cell-cycle regulation.MethodsUsing a haplotype-based analysis, this study aimed to investigate whether genetic polymorphisms in these four genes may contribute to ovarian cancer susceptibility. A total of 22 single nucleotide polymorphisms (SNPs) in these four genes were genotyped in 287 cases of ovarian serous cystadenocarcinomas and 618 age-matched cancer-free controls from the Chinese Han population, and then haplotype blocks were reconstructed according to our genotyping data and linkage disequilibrium (LD) status of these SNPs.ResultsFor AURKA, we found that haplotype GA [rs6064391 (T→G) + rs911162 (G→A)] was strongly associated with decreased ovarian cancer risk (adjusted OR = 0.31, 95% CI = 0.15–0.63, P = 0.0012). For BRCA1, we found that haplotype CGTAG was associated with decreased ovarian cancer risk (adjusted OR = 0.64, 95% CI = 0.41–0.98, P = 0.0417). Moreover, women harboring homozygous GA/CGTAG haplotypes showed the lowest risk (OR = 0.12, 95% CI = 0.02–0.94, P = 0.0438). In CCNE1, the SNPs rs3218035 and rs3218042 were significantly associated with increased ovarian cancer risk (rs3218035: adjusted OR = 5.20, 95% CI = 1.85–14.52, P = 0.0017; rs3218042: adjusted OR = 4.98, 95% CI = 1.75–14.19, P = 0.0027). For CDK2, no significant association was found.ConclusionsThis study indicates that genetic polymorphisms of AURKA, BRCA1 and CCNE1 may affect ovarian cancer susceptibility in Chinese Han women.     

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.     

Application of liquid chromatography–mass spectrometry to measure short chain fatty acids in blood

A new liquid chromatography–mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 μmol/l (R² ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 μmol/l for propionic and butyric acid and 0.1 μmol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples.     

Synthesis and anti-cancer activity of chalcone linked imidazolones

A series of novel chalcone linked imidazolones were prepared and evaluated for their anti-cancer activity against a panel of 53 human tumour cell lines derived from nine different cancer types: leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. Some of these hybrids (6, 7 and 8) showed good anti-cancer activity with GI₅₀ values ranging from 1.26 to 13.9 μM. When breast carcinoma cells (MCF-7) were treated with 10 μM concentration of compounds TMAC, CA-4, 6 and 8 cell cycle arrest was observed in G2/M phase. Surprisingly, the increased concentration of the same compound to 30 μM caused accumulation of cells in G0/G1 phase of the cell cycle.     

Effects of estradiol and progesterone on circulating LH and FSH secretion,and ovarian antral follicle growth in anestrous ewes

In the ewe, ovarian antral follicles emerge or grow in a wave-like pattern and each wave is preceded by a peak in the serum FSH level. The purpose of the current study was to investigate whether in anestrous Western White Face ewes, a combination of progesterone and estradiol affects the circulating FSH peak secretion and the number of small ovarian follicles. Five ewes were treated with subcutaneous silastic rubber implants (10 cm × 0.47 cm), containing 10% estradiol-17β w/w (controls) and 5 ewes were treated with the same estradiol implant, along with subcutaneous implants (11 cm × 0.48 cm) containing 10% progesterone w/w for 12 days. Daily transrectal ovarian ultrasonography and blood sampling was performed from 5 days before, to 9 days after the period of implantation. Blood samples were also taken every 12 min for a 6 h period on day −2, 6 and 13 prior to or after implant insertion (day 0, day of implant insertion). Pulsatility in the serum LH levels was eliminated by the implants (P < 0.05). During the implantation period, the serum FSH peak amplitude was lower in ewes treated with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol (P < 0.05). No follicular waves emerged during implant treatment in both groups (P < 0.05) and the number of serum FSH peaks did not differ during implantation, compared to before implantation. During the implantation period, the number of small follicles did not differ in ewes with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol. To conclude, supra-physiological concentrations of estradiol completely eliminated the serum LH pulsatality and suppressed the follicular wave emergence, while the FSH secretory peaks that preceded the follicular waves were not affected. Supra-physiological concentrations of estradiol-17β with physiological concentrations of progesterone decreased the serum FSH peak amplitude, eliminated the serum LH pulses, but did not decrease the size of the small follicle pool in anestrous ewes.     

Operation of an Anammox SBR in the presence of two broad-spectrum antibiotics

The feasibility of the anaerobic ammonium oxidation (Anammox) process to treat wastewaters containing antibiotics was studied in this work. Concentrations ranging from 100 to 1000 mg L^?1 for tetracycline hydrochloride and from 250 to 1000 mg L^?1 for chloramphenicol were tested in batch assays. A strong inhibitory effect was observed for both antibiotics.A concentration of 20 mg L^?1 of chloramphenicol was continuously added to an Anammox Sequential Batch Reactor (SBR) system, causing a decrease of the nitrogen removal efficiency of 25%. The Specific Anammox Activity (SAA) of the biomass also decreased from 0.25 to 0.05 g N (g VSS d)^?1. Similar effects were observed when 50 mg L^?1 of tetracycline hydrochloride were continuously fed. Both antibiotics did not cause any changes in the physical properties of the biomass. A previous degradation step could be necessary in order to treat wastewaters containing inhibitory concentrations of antibiotics by the Anammox process.     

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile–potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0–62.5 mg/L for tazobactam and 0–500 mg/L for piperacillin). The equation for regression line were y = 19x ? 1.4 for tazobactam and y = 1.7x ? 0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.     

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 human chorionic gonadotropin combined with clomiphene on Serum E2, FSH,LH and PRL levels in patients with polycystic ovarian syndrome

ObjectiveEffects of human chorionic gonadotropin combined with clomiphene on serum E₂, FSH, LH and PRL levels in patients with polycystic ovarian syndrome were analyzed.Methods90 patients with polycystic ovarian syndrome treated from January 2015 to March 2016 were randomly and evenly divided into control group and observation group. Patients in the control group were only treated with clomiphene. On the basis of the treatment in control group, human chorionic gonadotropin was added in the treatment of observation group. The changes of E₂, FSH, LH, PRL levels were compared between two groups before and after the treatment. Clinical curative effects of patients in the two groups was evaluated. Adverse reactions during treatment in two groups were observed and recorded. The incidence of adverse reactions was calculated.ResultsSerum E₂, FSH, LH and PRL levels in the two groups decreased significantly after treatment compared with that before treatment. The difference is statistical significant (P < 0.05). After the treatment, E₂, FSH, LH and PRL levels in the observation group were lower than that in the control group and the difference is statistical significant (P < 0.05). Total effective rate was 64.44% in the control group and 93.33% in the observation group. There were statistically significant difference in clinical curative effects in the two groups (P < 0.05). Different degrees of adverse reactions were found in both groups during treatment, such as nausea, vomiting, anorexia, liver dysfunction. There were 2 cases of nausea, 2 cases of vomiting, 3 cases of anorexia and 1 case of liver dysfunction from the 45 patients in control group. The total incidence of adverse reactions was 17.78% (8/45). There were 1 case of nausea, 1 case of vomiting, 1 case of anorexia and no liver dysfunction from the 45 patients in observation group. The total incidence of adverse reactions was 6.67% (3/45). The total incidence of adverse reactions in the observation group was significantly higher than that in the control group and the difference was not statistically significant (P > 0.05).ConclusionCombined use of human chorionic gonadotropin can significantly reduce serum E₂, FSH, LH and PRL levels, improve clinical curative effects and reduce the incidence of adverse reactions. Human chorionic gonadotropin has high application value on the treatment of polycystic ovary syndrome.     

Effects of dietary aluminum source and concentration on mineral status of feeder lambs

A 100 d experiment was conducted to determine the effects of aluminum (Al) source and concentration on mineral status, emphasizing phosphorus (P), of 50 feeder lambs. Six treatments, fed at 10% of the total diet, were formulated using two sources of Al, AlCl₃ and an Al-based water treatment residual (WTR, 11.1% Al), with varying levels of Al and P: (1) control (10% sand, C), (2) low WTR (2.5% WTR and 7.5% sand, L-WTR), (3) AlCl₃ with added P (1% AlCl₃, 9% sand, and 0.4% P, AlCl₃ + P), (4) high WTR (10% WTR, H-WTR), (5) AlCl₃ (1% AlCl₃ and 9% sand, AlCl₃), and (6) high WTR with added P (10% WTR and 0.4% P, H-WTR + P). The total Al varied from 0.037 to 1.2% among diets. Only lambs fed the high WTR diet without P supplementation (H-WTR) decreased feed intakes. These lambs consumed about half as much feed as lambs on all the other treatments, and had lower (P < 0.05) BW from d 84 on. Lambs receiving the H-WTR had the lowest bone Ca, P and Mg concentrations (fresh basis, mg/cm³) and lowest bone mineral content (BMC) as determined by radiographs (mm of Al). Results for the lambs on H-WTR were confounded by the greatly reduced feed intake of animals on this treatment. Plasma P decreased in all lambs consuming Al, regardless of Al source, but the effects were less severe in animals provided additional P supplementation (AlCl₃ + P and H-WTR + P). Apparent absorption of P was affected by concentration and source of Al in two metabolism trials (n = 42) beginning on d 34 and d 70, respectively. In the first trial, d 34, lambs receiving AlCl₃ treatment had reduced apparent P absorption, −17.7% (P < 0.05), when compared to all other treatments. In the d 70 trial, lambs receiving both AlCl₃ and H-WTR treatments were negatively impacted (P < 0.05) compared to the control, −20.9 and −2.5% apparent P absorption, respectively, but were no longer different from one another (P > 0.05). Diets containing 1.2% Al as WTR without P supplementation depressed feed intakes, weight gains, plasma P concentrations (P < 0.05), and BMC. However, given adequate P supplementation, even lambs consuming this amount of Al did not suffer detrimental effects, as lambs on H-WTR + P did not differ from the control (P > 0.05) in feed intakes, weight gains, or BMC.     

Effect of different dietary energy level intakes on efficiency of estrus synchronization and fertility in Mashona goat does

The objective of the study was to determine the effects of three dietary energy levels: 0.27 (low level: LL); 0.53 (medium level: ML), and 1.06 (high level: HL) MJ ME kg⁻¹ W^0.75 on estrus synchronization and fertility in Mashona goat does. Forty-five multiparous Mashona goat does of average bodyweight 19.9±2.5 kg were randomly allocated in equal numbers to the three dietary energy levels. The diets were made from a complete feed ration providing 9.83 MJ ME kg⁻¹ DM and 15.5% CP kg⁻¹ DM. Does were fed initially during a 60-day pre-synchronization period, and blood samples were collected twice a week for the determination of plasma progesterone concentrations to ascertain ovarian activity. Intramuscular injections of cloprostenol (100 μg each) were administered 11 days apart. Immediately after the second injection of cloprostenol, three fertile bucks were introduced to the does and were left with the does for 21 days. The does were maintained on their dietary treatments throughout gestation except for those does in the LL treatment. Pregnancy was diagnosed 90 days post-mating using an ultrasound scanner. After pregnancy diagnosis, does on the LL treatment were randomly allocated to ML (n=7) and HL (n=8) treatments. During the pre-synchronization period, does on the LL treatment lost 12.3% whereas those on ML and HL treatments gained 2.1 and 28.8% of their initial bodymasses, respectively. The proportion of does exhibiting overt estrus within 96 h after the last cloprostenol injection was significantly lower (P<0.05) for does on the LL treatment (60%) than for those on ML (93%) or HL (100%) treatments, respectively. However, based on plasma progesterone concentrations, the percentage of does on the LL treatment that exhibited ovarian cycles was numerically lower than that of does that were bred (40 versus 73%). Conception, fecundity and twinning rates were significantly lower (P<0.05) on the LL treatment than on the ML and HL treatments. These results indicate that feeding Mashona goat does 0.27 MJ ME kg⁻¹ W^0.75 compared to 0.53 and 1.06 MJ ME kg⁻¹ W^0.75 reduces the expression of estrus, conception, fecundity and twinning rates, and that feeding 0.53 MJ ME kg⁻¹ W^0.75 suffices for optimum reproduction. In addition, the results suggest that cloprostenol administration may induce ovarian cycles in reproductively quiescent does on dietary energy restriction.     

Seasonal variation in tissue estrogen-2/4-hydroxylases (EH) and in vitro effects of steroids on ovarian EH activity in the catfish Heteropneustes fossilis

A radiometric assay was used to measure microsomal EH activity from tritiated H₂O formed during the conversion of [2,4 ³H] estradiol-17β into catecholestrogens in the microsomal fractions of liver, brain and ovary of the catfish Heteropneustes fossilis. The validation data show that enzyme activity increased with incubation time, and substrate and cofactor (NADPH) concentrations, elicited temperature optima of 30–37 °C and pH optima of 6.8–7.8. EH activity was strongly NADPH-dependent and in its absence only 13.48% activity was recorded. Liver recorded the highest enzyme activity, followed by brain and ovary. EH activity showed a significant seasonal variation with the peak activity in spawning phase and the lowest activity in resting phase. In the ovary, the follicular layer (theca and granulosa) elicited the highest activity over that of the denuded oocytes. Modulatory effects of steroids on ovarian enzyme activity were further demonstrated. The incubation of postvitellogenic follicles with 1, 10 or 100 nM concentrations of various steroids for 24 h produced varied effects on EH activity. Progesterone and 2-hydroxyestradiol-17β elicited strong suppressive effects on enzyme activity. Estrogens (E₁, E₂ and E₃) suppressed the activity in a concentration-dependent manner. Among the progestins tested, 17,20α-dihydroxy-4-pregnen-3-one, the isomer of 17,20β-dihydroxy-4-pregnen-3-one (a teleost maturation-inducing steroid) showed the lowest depressing effect. Among androgens, the testosterone metabolite 11-ketotestosterone (functional teleost androgen) showed a high suppressing effect. Corticosteroids elicited low activity with cortisol suppressed the activity at higher concentrations. The study will form a basis to understand the physiological role of catecholestrogens in ovarian functions.     

Cadmium,lead and mercury concentrations and their influence on morphological parameters in blood donors from different age groups from southern Poland

Due to industrial development, environmental contamination with metals increases which leads to higher human exposure via air, water and food. In order to evaluate the level of the present exposition, the concentrations of metals can be measured in such biological materials as human blood. In this study, we assessed the concentrations of cadmium (Cd), mercury (Hg) and lead (Pb) in blood samples from male blood donors from southern Poland (Europe) born in 1994 (n = 30) and between 1947 and 1955 (n = 30). Higher levels of Pb were seen in the group of older men (4.48 vs 2.48 μg/L), whereas the Hg levels were lower (1.78 vs 4.28 μg/L). Cd concentrations did not differ between age groups (0.56 μg/L). The levels of Cd and Pb in older donors were significantly correlated (Spearman R 0.5135). We also observed a positive correlation between the number of red blood cells (RBC) and Hg concentrations in the older group (Spearman R 0.4271). Additionally, we noted numerous correlations among morphological parameters. Based on our results, we can state that metals influence the blood morphology and their concentrations in blood vary among age groups.     

C-11 diamino cryptolepine derivatives NSC748392, NSC748393, and NSC748394: Anticancer profile and G-quadruplex stabilization

G-Quadruplex DNA ligands are promising novel anticancer agents with potentially fewer side effects and greater selectivity than standard anticancer drugs. However, the design of G-quadruplex ligands remains challenging since known chemical features increasing selectivity have often compromised drugability. Three C-11 diamino cryptolepine derivatives, with significant chemical differences between the side chains, low cytotoxicity to mammalian non-tumor cells (Vero cells) and drug-like properties, were selected for anticancer drug screening in the NCI Developmental Therapeutics Program. The three compounds showed good in vitro anticancer profiles with GI₅₀ averages at sub-micromolar concentrations (0.32–0.78 μM), cytostatic effects (TGI) at micromolar concentrations (1.3–6.9 μM) and moderate cytotoxic effects to cancer cells (LC₅₀) also at micromolar concentrations (4.7–33 μM), but only the compound with a linear alkylamine side chain (NSC748393) showed a good score in the in vivo anticancer Hollow Fiber assay. compare analysis of growth inhibition profile of NSC748393 suggested a multi-target mechanism. G-Quadruplex DNA binding affinity and selectivity studies by FRET-melting assays showed that NSC748392 and NSC478393, with aliphatic amine side chains, are good G-quadruplex ligands but not selective, whereas a C-11 aromatic side chain, as in NSC748394, increases selectivity although with decreasing binding affinity. Overall, NSC748393 can be considered a lead molecule for the design of effective but more selective anticancer drugs targeting telomeric G-quadruplexes.     

Toxicokinetics of di(2-ethylhexyl) phthalate (DEHP) and its effects on luteal function in sheep

The aim of the present study was to determine the toxicokinetics of short-term exposures to di(2-ethylhexyl) phthalate (DEHP) and its effects on ovarian cyclicity and luteal function using a sheep experimental model. For establishing the model, we examined the clearance of DEHP after intravenous (i.v.) and intramuscular (i.m.) administration of a single dose of 25 mg/kg body weight (b.w.) and after i.m. administration of two different doses (25 and 50 mg/kg b.w.; DEHP25 and DEHP50, respectively) three times a week for two months. Results showed a significant, dose-dependent effect of DEHP administration, when compared to the control group (CTL; untreated ewes; n = 6), on the duration of the ewes’ estrous cycles (17.1 ± 0.5 days, CTL; 15.1 ± 0.9 days, DEHP25; 12.0 ± 0.8 days, DEHP50; p < 0.05); 94.9% of the cycles were of regular duration (15–19 days) in CTL, but only 51.1% and 25.4% in DEHP25 and DEHP50, respectively. Corpora lutea (CL) were smaller in DEHP50 than in DEHP25 (p < 0.05) and were smaller in both groups than in CTL (p < 0.005), but the maximum plasma concentrations of progesterone were greater (p < 0.05) in DEHP25 and DEHP50 than in CTL. In conclusion, the exposure of cycling ewes to DEHP causes shortening of the ovulatory cycles due mainly to a reduction in the size and lifespan of CL. However, the exposure to the phthalate is also associated with an increase in circulating concentrations of progesterone, suggesting the influence of DEHP on steroid metabolism.     

Optimization of a rapid microwave-assisted extraction method for the simultaneous determination of opiates,cocaine and their metabolites in human hair

A rapid and cleanup-free microwave-assisted extraction (MAE) method is proposed for the simultaneous extraction of six illegal drugs of abuse – cocaine, benzoylecgonine (BZE), cocaethylene (CCE), morphine, 6-monoacethylmorphine (6AM) and codeine – from human hair samples. The analytes were determined using high performance liquid chromatography (HPLC) with photodiode array UV detection. The influence of several variables on the efficiency of the MAE procedure was investigated in detail by a multi-objective optimization approach based on a hybrid experimental design (17 experiments) and desirability functions. Six drugs were successfully extracted from human hair with recoveries close to 100% and good reproducibility (<3.6% RSD) under the optimal MAE conditions: 11 mL dichloromethane (DCM) extraction solvent, 60 °C extraction temperature, 9 min extraction time and 0.5 mL of methanol (MeOH) added to 50 mg of the hair sample in the extraction vessels. Limits of quantification of 0.2 ng mg^?1 were found for the studied compounds. A comparison of sample preparation procedures, including MAE, enzymatic digestion and digestion by aqueous acids, was also conducted. The results indicated that the global behaviour of sample procedures provided similar satisfactory recoveries ranging from 86 to 100%. Indeed, the MAE procedure resulted in a reduction of extraction time by 100-fold and the elimination of cleanup steps. Slightly higher recoveries of morphine, 6AM, BZE and CCE, at 1 ng mg^?1 concentration level and cocaine at 40 ng mg^?1 concentration level, were achieved using MAE. Lastly, the proposed MAE method was applied to several human hair samples from multidrug abusers.