Urea derivative MTU improves stress tolerance and yield in wheat by promoting cyclic electron flow around PSI.

Increasing crop productivity under optimal conditions and mitigating yield losses under stressful conditions is a major challenge in contemporary agriculture. We have recently identified an effective anti-senescence compound (MTU, [1-(2-methoxyethyl)-3-(1,2,3-thiadiazol-5yl)urea]) in in vitro studies. Here, we show that MTU delayed both age- and stress-induced senescence of wheat plants (Triticum aestivum L.) by enhancing the abundance of PSI supercomplex with LHCa antennae (PSI-LHCa) and promoting the cyclic electron flow (CEF) around PSI. We suppose that this rarely-observed phenomenon blocks the disintegration of photosynthetic apparatus and maintains its activity as was reflected by the faster growth rate of wheat in optimal conditions and under drought and heat stress. Our multiyear field trial analysis further shows that the treatment with 0.4 g ha-1 of MTU enhanced average grain yields of field-grown wheat and barley (Hordeum vulgare L.) by 5-8%. Interestingly, the analysis of gene expression and hormone profiling confirms that MTU acts without the involvement of cytokinins or other phytohormones. Moreover, MTU appears to be the only chemical reported to date to affect PSI stability and activity. Our results indicate a central role of PSI and CEF in the onset of senescence with implications in yield management at least for cereal species.

Effect of mercury on porcine ovarian granulosa cells in vitro.

The objective of this in vitro study was to examine dose-dependent changes in the secretion activity [progesterone (P4) and insulin-like growth factor-I (IGF-I)] of porcine ovarian granulosa cells after experimental mercury (Hg) administration, including its apoptotic potential so as to ascertain the possible involvement of Hg in steroidogenesis. Ovarian granulosa cells were incubated with mercuric chloride [mercury (II) chloride or HgCl2] at the doses 50-250 µg mL(-1) for 18 h and compared with control group without Hg addition. Release of P4 and IGF-I by ovarian granulosa cells was assessed by RIA and apoptosis by TUNEL assay. Observations show that P4 release by granulosa cells was significantly (P < 0.05) inhibited at all the doses, while IGF-I release was not affected at any of the doses used, although a decreasing trend in the release of IGF-I was noted in comparison to control. An increasing trend of apoptosis of granulosa cells was noted, the difference being significant (P < 0.05) only at the dose 130 µg mL(-1) HgCl2, in comparison to control. Obtained data suggest a direct effect of Hg on the release of steroid hormone progesterone but not growth factor IGF-I, and a dose-dependent effect on apoptosis of porcine ovarian granulosa cells. Results indicate the interference of Hg in the pathways of steroidogenesis and apoptosis of porcine ovarian granulosa cells.

Effects of mercury on the steroidogenesis of human adrenocarcinoma (NCI-H295R) cell line.

In this study the NCI-H295R human adrenocortical carcinoma cell line was used as an in vitro biological model to study the effect of mercury (HgCl(2)) on the steroidogenesis. The cells were cultured for 48 h with addition of 1.0; 5.0; 25; 50 or 100 µM of HgCl(2) and compared to control. Cell viability was measured by the MTT (metabolic activity) assay estimation for the mitochondria structural integrity. Quantification of testosterone and progesterone directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Concentration-dependent depression in testosterone production was detected particularly for higher concentration of Hg(2+). Progesterone production was also decreased, but at the lower concentrations (1.0 and 5.0 µM) of Hg(2+) this decline was lower compared to depression of testosterone. The cell viability significantly decreased at 25 µM and higher concentration of Hg(2+). However, at 25 µM Hg(2+) exposure the cell viability remained relatively high (> 80%). Results of the study indicate dose-dependent decreases in both testosterone and progesterone production of H295R cell culture following a 48 h in vitro HgCl(2) exposure. The results suggest that Hg has detrimental effects on steroid hormone synthesis also at very low concentrations and consecutively on reproductive physiology.

In vitro study on the effects of lead and mercury on porcine ovarian granulosa cells.

The heavy metals lead (Pb) and mercury (Hg) pose potential risks to sustainability of environment and thus to our future generations. General objective of this in vitro study was to examine the secretory activity of porcine ovarian granulosa cells after Pb and Hg administration and to outline the potential intracellular mediators of its effects. For this purpose, release of insulin-like growth factor I (IGF-I) and steroid hormone progesterone (P(4)), expression of proliferation- related (cyclin B1) and apoptosis-related (caspase-3) peptides was examined in porcine ovarian granulosa cells after heavy metals administration. Obtained data indicate Pb-induced inhibition of IGF-I release at lower doses (0.063 mg/mL and 0.046 mg/mL) by ovarian granulosa cells. However, P(4) release was not influenced by Pb addition, while the expression of cyclin B1 and caspase-3 was induced by Pb addition. These results indicate that Pb can affect the pathway of proliferation and apoptosis of porcine ovarian granulosa cells through intracellular substances such as cyclin B1 and caspase-3. On the other hand, the P(4) release by ovarian granulosa cells of pregnant gilts was stimulated by experimental Pb administration at doses of 0.25 mg/mL and 0.063 mg/mL and experimental Hg administration at doses 0.25 mg/mL and 0.083 mg/mL. P(4) release by ovarian cells of pregnant gilts was not influenced by a combinatory dose of FSH (1.0 ng/mL) + Pb (0.083 mg/mL) + Hg (0.083 mg/mL) but it was inhibited by experimental administration of FSH (10 ng/mL) + Pb (0.25 ng/mL) + Hg (0.25 ng/mL). Possible involvement of heavy metals - Pb and Hg and pituitary hormone FSH, in the regulation of P(4) release by porcine ovarian granulosa cells of pregnant gilts was noted. Data obtained from in vitro studies suggest the dose dependent association of heavy metals administration with the hormonal release by porcine ovarian granulosa cells. This association also depended on pregnancy of the gilts.

In vitro toxicity of mercuric chloride on rabbit spermatozoa motility and cell membrane integrity.

In this in vitro study the effects of mercuric chloride on the motility and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated using CASA method and Annexin analysis was used for detection of structural changes. The concentration of mercury in the medium varied from 5.0 to 83.3 microg HgCl(2)/mL. At Time 0 the highest motility was detected in the control group (67.09 +/- 8.72%). Motility in groups with mercury administration was lower in comparison with control. Significant differences were detected in groups with 50.0-83.3 microg HgCl(2)/mL (P < 0.001) at Time 0. After 60 and 120 minutes of incubation with mercuric chloride the motility significantly decreased almost in all experimental groups. Progressive motility had a decreasing trend in all experimental groups. At time 60 and 120 significant differences were noted in the group receiving 6.25-83.3 microg HgCl(2)/mL. Significant differences were detected in all experimental groups, except the group with the lowest mercuric chloride administration. The concentration-dependent decrease of spermatozoa progressive motility up to 50% of control was detected for groups receiving 50.0 - 83.3 microg HgCl(2)/mL at Time 0, for groups receiving 12.5-83.3 microg HgCl(2)/mL at Time 60 and 120, decreasing from 36.46 +/- 18.73% to 1.03 +/- 2.50%. Detailed evaluation of spermatozoa distance (DAP, DCL, and DSL) and velocity (VAP, VCL, and VSL) parameters as well as straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of spermatozoa revealed decrease in groups with the highest mercury concentration in comparison with the control group at all time periods. Detection of spermatozoa with disordered membrane was carried out for groups with higher mercury concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the mercury exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In mercury-exposed groups positive reaction proved alteration in anterial part of head (acrosome), connection part (connection piece) and in mitochondrial segment. Detected data evidently confirm adverse effects of high mercuric chloride concentrations in rabbit semen on spermatozoa motility parameters.

Concentration of trace elements in human semen and relation to spermatozoa quality.

In this study the concentrations of trace elements such as lead, cadmium, iron, nickel, copper and zinc in the human semen (n = 47), occurrence of pathological spermatozoa, and correlations of these elements to pathological forms were investigated. For each sample of human spermatozoa at least 500 spermatozoa were evaluated. Metal contents were determined by the voltametric method and flame absorption spectrophotometry method. The concentrations of trace elements in human semen were: lead 1.49 +/- 0.40 mg x kg(- 1), cadmium 0.13 +/- 0.15 mg x kg(- 1), iron 2.59 +/- 0.21 mg x kg(- 1), nickel 0.40 +/- 0.07 mg x kg(- 1), copper 0.28 +/- 0.06 mg x kg(- 1), and zinc 153.93 +/- 67.08 mg x kg(- 1), respectively. The total percentage of pathological spermatozoa was 41.61 +/- 9.80% with predominancy of broken flagellum, flagellum torso and separated flagellum. In relation to trace elements the analysis showed correlation between copper and lead (r = -0.47), nickel and iron (r = 0.36), lead and flagellum ball (r = -0.39), cadmium and large heads (r = 0.37) and between iron and other forms of pathological spermatozoa (r = -0.32). Results of this study describe possible effects of trace elements on the spermatozoa quality in normal human sperm.

Mercury-induced alterations in rat kidneys and testes in vivo.

In this study effects of mercury administration on the kidney and testicular structure of adult rats were evaluated. Rats received mercury (HgCl2) in single intraperitoneal dose 20 mg HgCl2 (group A), 10 mg HgCl2 (group B) and 5 mg HgCl2 (group C) per kilogram of body weight and were killed after 48 hours following mercury administration. After the preparation of histological samples the results were compared with control group (K). In kidney decreased diameters of glomeruli and renal corpuscles, damaged tubules with affected quality of tubular cells and infiltration of interstitium were detected. Quantitative analysis demonstrated increased relative volume of tubules and renal corpuscles. Also the number of nuclei and glomeruli was increased in all experimental groups. The diameter of glomeruli and renal corpuscles was decreased. In testis undulation of basal membrane, dilatation of blood vessels in interstitium and occurrence of empty spaces in germinal epithelium were observed. Decreased relative volume of germinal epithelium, increased relative volume of interstitium and increased apoptosis occurrence suggest damaged interstitium and revealed occurrence of edemas. The relative volume of seminiferous tubules showed higher luminization. The number of nuclei was decreased in all experimental groups what is in positive relation with occurrence of empty spaces. Also other evaluated criteria demonstrated significant differences between control group and experimental groups. This study reports a negative effect of mercury on the structure and function of kidney and testes.