The key biological active molecule of soya is the isoflavone daidzein, which possesses phytoestrogenic activity. The direct effect of soya and daidzein on ovarian cell functions is not known. This study examined the effect of daidzein on basic porcine ovarian granulosa cell functions and the response to follicle-stimulating hormone (FSH). We studied the effects of daidzein (0, 1, 10 and 100 μm), FSH (0, 0.01, 0.1, 1 IU/ml) and combinations of FSH (0, 0.01, 0.1, 1 IU/ml) + daidzein (50 μm) on proliferation, apoptosis and hormone release from cultured porcine ovarian granulosa cells and ovarian follicles. The expression of a proliferation-related peptide (PCNA) and an apoptosis-related peptide (Bax) was analysed using immunocytochemistry. The release of progesterone (P4) and testosterone (T) was detected using EIA. Leptin output was analysed using RIA. Daidzein administration increased granulosa cell proliferation, apoptosis and T and leptin release but inhibited P4 output. Daidzein also increased T release and decreased P4 release from cultured ovarian follicles. Follicle-stimulating hormone stimulated granulosa cell proliferation, apoptosis and P4, T and leptin release. The addition of daidzein promoted FSH-stimulated apoptosis (but not proliferation) but suppressed FSH-stimulated P4, T and leptin release. Our observations of FSH action confirm previous data on the stimulatory effect of FSH on ovarian cell proliferation, apoptosis and steroidogenesis and demonstrate for the first time the involvement of FSH in the upregulation of ovarian leptin release. Our observations of daidzein effects demonstrated for the first time that this soya isoflavone affected basic ovarian cell functions (proliferation, apoptosis and hormones release) and modified the effects of FSH. Daidzein promoted FSH action on ovarian cell proliferation and apoptosis and suppressed, and even inverted, FSH action on hormone release. The direct action of daidzein on basic ovarian cell functions and the ability of these cells to respond to FSH indicate the potential influence of soya-containing diets on female reproductive processes via direct action on the ovary.

A case of suspected acute and lethal intoxication caused by colchicine has been reported. The woman was hospitalized after her suspicion of suicidal poisoning by a rare autumn crocus (Colchicum autumnale). Suspected colchicine poisoning was confirmed using a novel UHPLC method with a modern reversed‐phase stationary phase with a sub 2‐micron superficial porous particle size combined with a QTOF mass spectrometer. Sample preparation procedure included the addition of propiverine as internal standard, protein precipitation using methanol and solid phase extraction. High‐resolution MS only and targeted MS/MS modes are reported for the qualitative analysis and screening of other potential drugs of abuse in blood samples. All Ion MS mode was used for quantitative determination of colchicine afterward. The concentration of colchicine in the blood sample was approximately 41 ng/mL, and more than 200 μg/mL of the plant extract used for the suicide.

Heavy metals cause changes either in the pattern of cellular stress protein expression or in the enzyme activity. In the present study, the effect of three different toxic element treatments (heavy metals Pb, Cd and metaloid As) on small heat shock proteins synthesis as well as the superoxide dismutase (SOD) activity in root tips of yellow lupine (Lupinus luteus L., varieta Juno) was studied. Solutions with four different concentrations from each salt [Pb(NO 3 ) 2 , Cd(NO 3 ) 2 and As 2 O 3 ] were applied to achieve ascending oxidative stress conditions in comparison to unstressed, water treated, control variant. SDS-PAGE of crude protein extracts from treated variants showed enhanced protein signals in 15-17 kDa area comparing to the control one. The increased proteosynthetic activity indicates the possible participation of the low molecular weight proteins in the cell defence reactions. Similar increasing tendency was observed in the case of SOD enzymatic activity. Presence of heavy metals resulted in immediate elevated enzymatic response. Our results showed that methods applied in this study can be used for detection of oxidative stress in plants.

The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed at 0.5, 1, 2, 3, 4, 8, 12, and 16 h postactivation (hpa). Parthenogenetic (PA) embryos were used as control. The SCNT and PA embryos were processed for lacmoid staining, autoradiography, transmission electron microscopy (TEM), and immunofluorescence localization of: upstream binding factor (UBF) and fibrillarin at 4 and 12 hpa. Likewise, starved and nonstarved fibroblasts were processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillogranular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. In conclusion, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development.

No abstract

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). Bovine and porcine intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures in sequence-specific interactions between the ooplasm and chromatin of another genus. In conclusion, the results demonstrate a possible reason why the intergeneric SCNT embryos never reached the full term.

Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.