Mitochondrial patterns in bovine oocytes with different meiotic competence related to their in vitro maturation.

This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.

Characteristics of bovine oocytes with different meiotic competence in terms of their mitochondrial status and expression of nuclear-encoded factors.

The present study was designed to characterize bovine oocytes with different meiotic competence and atresia levels in terms of their mitochondrial status. Oocyte subpopulations were recovered either from medium (MF) or small (SF) follicles and categorized as healthy, light-atretic and mid-atretic according to oocyte morphology. Mitochondrial activity, morphology and distribution, adenosine triphosphate (ATP) content and expression of mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1) were assessed before (GV) and after (MII) maturation. The data were related to follicular size regardless of or with regard to oocyte atresia. Regardless of atresia, the MF subpopulation showed a significantly higher mitochondrial activity and frequency of oocytes with granulated mitochondria at GV and clustered mitochondria at MII than the SF subpopulation. With regard to atresia, mitochondrial activity decreased from healthy to mid-atretic oocytes in both MF and SF subpopulations at GV, but in the SF subpopulation at MII, the mitochondrial activity and frequency of oocytes with clustered mitochondria were significantly higher in light-atretic than in healthy oocytes. The light-atretic oocytes also produced more ATP than healthy ones in both SF and MF subpopulations. However, a significantly higher relative abundance of mRNA TFAM was found in SF than MF subpopulations at GV, and this difference remained in mid-atretic oocytes at MII. It can be concluded that meiotic competence and atresia level influence mitochondrial status of immature bovine oocytes. After maturation, healthy oocytes from medium follicles and light-atretic oocytes from small follicles were more developed in terms of mitochondrial status than the other oocytes.

Transverse spin forces and non-equilibrium particle dynamics in a circularly polarized vacuum optical trap.

We provide a vivid demonstration of the mechanical effect of transverse spin momentum in an optical beam in free space. This component of the Poynting momentum was previously thought to be virtual, and unmeasurable. Here, its effect is revealed in the inertial motion of a probe particle in a circularly polarized Gaussian trap, in vacuum. Transverse spin forces combine with thermal fluctuations to induce a striking range of non-equilibrium phenomena. With increasing beam power we observe (i) growing departures from energy equipartition, (ii) the formation of coherent, thermally excited orbits and, ultimately, (iii) the ejection of the particle from the trap. As well as corroborating existing measurements of spin momentum, our results reveal its dynamic effect. We show how the under-damped motion of probe particles in structured light fields can expose the nature and morphology of optical momentum flows, and provide a testbed for elementary non-equilibrium statistical mechanics.

A reversible block at the G1/S border during cell cycle progression of mouse embryos.

Late 2-cell stage mouse embryos were cultured in M-199 plus 100 micrograms/ml Na pyruvate 25 micrograms/ml gentamycin and 0.3% BSA with or without mimosine (200 microM, 150 microM, 100 microM and 50 microM) for a short (4-5 h) or long (18-20 h) culture period; after drug removal subsequent embryo development was evaluated. Late 2-cell stage mouse embryos treated with mimosine were blocked at the 4-cell stage. Autoradiographic studies show that mimosine inhibits cell cycle progression in mouse embryos at the G1/S boundary. The onset of DNA replication occurs within 15 min of releasing the embryos from mimosine block. Embryos pretreated with mimosine at 200 microM and 150 microM for 4-5 h progress after 3-4 days in culture to hatched blastocyst (71% and 79%, respectively) compared with control (90%). However a longer pretreatment (18-20 h) with mimosine at 200 microM was significantly detrimental to the subsequent developmental progression to hatched blastocyst (2% vs 81%, p < or = 0.05); the proportion of degenerated embryos was significantly increased with mimosine at 200 microM and 150 microM compared with control (57% and 28% vs 4%, p < or = 0.05) after 3-4 days in culture. Preliminary studies with mimosine treatment at 100 microM and 50 microM for 18-20 h show that 70% and 37% of the embryos were blocked at 4-cell stage, respectively. These results indicate that mimosine inhibits cell cycle progress in mouse embryos at the G1/S border and thus induces a reversible arrest in a dose- and time-dependent manner.

Retrieval of the pulse amplitude and phase from cross-phase modulation spectrograms using the simulated annealing method.

The simulated annealing method is used for retrieving the amplitude and phase from cross-phase modulation spectrograms. The method allows us to take into account the birefringence of the measurement fiber and resolution of the optical spectrum analyzer. The influence of the birefringence and analyzer resolution are discussed.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Genome reactivation in developing early pig embryos: an ultrastructural and autoradiographic analysis.

The onset of RNA synthesis in developing early pig embryos from 1-cell to 8-cell and morula stages was studied using high-resolution autoradiography of (5-3H)uridine incorporation. No transcriptional activity was detected in nuclei of 1- and 2-cell stage embryos with this technique. In these embryos nucleolus-like bodies (NLB) consist of sharply delineated, round, electron dense fibrillar masses. In the 4-cell stage embryos, the first uridine-3H incorporation in the nucleoplasm was detected and localized mainly near the regions of condensed chromatin. The first signs of reticulation and chromatin association were observed at the periphery of NLBs. In the next cell cycle (5- to 8-cell embryos) uridine-3H labelling was detected in the nucleoplasm and nucleoli. In these embryos, nucleoli consist of a central dense fibrillar mass without any transcriptional activity and fibrillo-granular cortex over which label was localized. The degree of functional restructure of nucleoli was variable within one blastomere or among different blastomeres, some nucleoli being more reticulated and showing more transcriptional activity than others. Fully developed nucleoli were present in early morulae. Electron dense unidentified structures described here as small dense round-shaped bodies (RDB) often surrounded by blocks of large chromatin granules were observed in intact 2-cell and alpha-amanitin treated 4-cell stage embryos. These structures did not show any transcriptional activity.

Chromosome condensation activity (CCA) in bisected C57BL/6JxCBA mouse oocytes.

Chromosome condensation activity (CCA) has been analysed in C57BL/6Jx CBA mouse oocytes bisected (i) shortly after germinal vesicle breakdown (GVBD), (ii) in metaphase I (MI) and (iii) in metaphase II (MII) into two equal halves (nucleated, enucleated) which were thereafter fused to S- or G2-phase 4-cell-stage mouse blastomeres. In nucleated halves, premature chromosome condensation (PCC) in transplanted nuclei was always induced irrespective of the cell cycle stage of the blastomere, whereas in enucleated halves only G2 nuclei underwent PCC after transplantation. Premature chromosome condensation in S-phase nuclei was induced only in enucleated halves produced shortly after GVBD. Although S-phase nuclei transplanted to MI or MII enucleated halves remained intact, their capacity to synthesize DNA was invariably suppressed. When spindles were destroyed by preincubation of the oocytes in colcemid before bisection, both nucleated and enucleated halves produced at MI or MII induced PCC of both G2- or S-phase nuclei. These results demonstrate that chromosome condensation activity in mammalian oocytes is compartmentalized rather than uniformly distributed across the cell, and that the enucleation of mammalian oocytes before nuclear transplantation may, under some conditions, influence the levels of CCA and subsequent response of introduced nuclei to cytoplasmic factors.

Immunological response of patients with tumors of the urogenital tract: a follow-up of cell-mediated antitumor immunity by the LAI test.

Antigen-induced inhibition of leukocyte adherence is a suitable method providing information on immunological response of the tumor bearers. Leukocytes from patients' blood were tested by an in vitro method to detect cell-mediated antitumor immunity. Positive reactions of leukocytes to the specific antigen were found in carcinoma of the uterine cervix, vulva and ovary in 13 patients. The modified LAI test evaluated on the basis of free cells in Eagle's medium using the specific antigen is a simplified reversed method which, however, keeps the specificity.

Culture of bovine oocytes from small antral follicles in meiosis-inhibiting medium with butyrolactone I: RNA synthesis, nucleolar morphology and meiotic competence.

Bovine oocytes originated from follicles of two different size categories (medium (M), 3-6 mm and small (S), 1-2 mm) were cultured for 24 and 70 h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100 microM butyrolactone I (BL I). At the end of culture, cumulus oocyte complexes (COC) from M and S follicles were labeled by 3H-uridine for 30 min. The autoradiography (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (4+,3+) of the GV. (2) When the COC were labeled after 24 and 70 h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (3+,2+) after 24 h and negligible labeling after 70 h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70 h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24 h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70 h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48 h. The subsequent 24 h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins directly after isolation, reached M II after 48 or 72 h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.

Gene expression and chromatin structure in the pre-implantation embryo.

The pre-implantation period of mammalian development includes the formation of the zygote, the activation of the embryonic genome (EGA), and the beginning of cellular differentiation. During this period, protamines are replaced by histones, the methylated haploid parental genomes undergo demethylation following formation of the diploid zygote, and maternal control of development is succeeded by zygotic control. Superimposed on this activation of the embryonic genome is the formation of a chromatin-mediated transcriptionally repressive state requiring enhancers for efficient gene expression. The development of this transcriptionally repressive state most likely occurs at the level of chromatin structure, because inducing histone hyperacetylation relieves the requirements for enhancers. Characterization of zygotic mRNA expression patterns during the pre-implantation period and their relationship to successful development in vitro and in vivo will be essential for defining optimized culture conditions and nuclear transfer protocols. The focus of this review is to summarize recent advances in this field and to discuss their implications for developmental biology.

Identification of seroreactive epitopes of human papillomavirus type 18 E7 protein by synthetic peptides.

Nine everlapping peptides covering the entire sequence of early protein E7 of human papillomavirus type 18 (HPV-18) were synthesized and tested as antigens with pools of selected human sera in ELISA. Peptides denoted 18/E7-2, 18/E7-3, and 18/E7-5 (amino acid positions 11-33, 21-40, and 41-60, respectively) were reactive with pooled sera originating from HPV-18 DNA-positive cervical cancer patients but not with sera from HPV-16 DNA-positive cervical cancer patients or from condyloma acuminata patients. This suggested that the epitopes contained in these peptides were HPV-18 type-specific, relative to HPV types 16, 11, and 6. On the other hand, 18/E7-1 (aa 1-23) and 18/E7-6 (aa 51-70) peptides were cross-reactive. The prevalence of antibodies reactive with 18/E7-2, 18/E7-3, and 18/E7-5 peptides in cervical carcinoma patients was very low. Thus, the utilization of these peptides for monitoring HPV-18 infection seems to be rather limited.

Pulpal response to adhesive resin systems applied to acid-etched vital dentin: damp versus dry primer application.

A number of studies have reported that acid etching of dentin is toxic to the cells of the odontoblastic layer and dental pulp. Other studies report that pulpal inflammation is a consequence of bacterial microleakage. The purpose of this study was to observe the degree of pulpal healing after pretreatment of vital dentin prior to placement of All-Bond and Scotch-bond 2 composite resin adhesives. Zinc oxide-eugenol cement and an acidic cement were employed as controls. One hundred twelve Class V nonexposed cavity preparations were placed throughout the dentitions of five healthy adult rhesus monkeys and observed at 3, 25, and 80 days. Various dentinal pretreatment procedures were employed. The All-Bond Universal primer system was placed on air-dried vital dentin in 23 cavities and on damp vital dentin in 27 cavities. Scotchbond 2 was placed as per manufacturer's instructions. All treatment procedures, materials, and times were represented in all animals. Placement of silicate cement resulted in the most severe pulpal responses at all time periods. Stained bacterial profiles in the remaining dentin on the axial walls of inflamed control pulps were associated with severe pulpal inflammation. These results indicate that acid etching of vital dentin does not impair pulpal healing in deep Class V cavities.

RNA synthesis in porcine blastomere nuclei introduced into in vitro matured ooplasm.

The objective was to investigate the RNA synthesis in porcine blastomere nuclei upon transplantation into in vitro matured enucleated oocytes. Nuclei from 2- to 8-cell porcine embryos were introduced into the ooplasm of in vitro matured and enucleated porcine oocytes by electrofusion, and the resultant reconstructed embryos were cultured in vitro. Before fusion or at different intervals after this event embryos were incubated with [3H]-uridine, fixed, and histologically processed for autoradiography in order to detect RNA synthesis. About two thirds of the embryos were considered to depict normal development. All blastomeres displayed pronounced RNA synthesis before fusion, at 3 and 9 h after fusion the synthesis decreased or ceased, and at 24-49 h some embryos resumed synthesis at the 1- to 2-cell stage.

[Potential carcinogenic factors in cervical intraepithelial neoplasia]. / Studie potenciálních karcinogenních faktoru u cervikálních intraepitelových neoplazií.

In a group of 22 women with cervical intraepithelial neoplasia and carcinoma of the cervix uteri and in a control group of 37 healthy women, a number of anamnestic and objective data about factors influencing these women and about consequences of such influence were analysed. Statistically significant differences between patients and controls were found in the character of work performed (physical vs. intellectual, p = 0.0312), the presence of stressing factors at the workplace (p = 0.02), a higher number of consumed meals per day in the control group (p = 0.0009), daily consumption of beer in a higher percentage of patients (p = 0.0015), the frequency of sexual intercourse in the last 2-3 months before the diagnosis of disease or examination (higher in controls, p = 0.0164) and subjective rating of sexual life in the fourth decade (better rating in controls, p = 0.0005). No differences were found between patients and controls in the number of sexual partners, the presence of antibodies against HSV-2 and other factors. According to data presented and data in the literature, external environment, some of the characteristics of sexual life and particularly infection with certain types of papillomaviruses have a certain influence on the development of cervical neoplasias.

[Correlation of clinical and virologic diagnosis of cervical infection with human papillomaviruses]. / Korelace klinické a virologické diagnostiky infekce cervixu lidskými papilomaviry.

The presented investigation is concerned with contemporary diagnostic possibilities of HPV Infection of the Cervix. The authors present the results of virological examinations of 228 female patients in the Centre for Oncological Prevention. The examination was made by hybridization techniques, using probes specific for HPV 6, 11, 16 and 18 and by serological methods where IgG antibodies were assessed against synthetic peptides, corresponding to several HPV epitopes, as antigens. 156 women (68.4%) were virologically positive, 72 (31.6%) were negative. Subsequently the authors investigated the diagnostic accuracy of HPV changes of the cervix by clinical methods, i.e. colposcopy and cytology, as compared with virological methods. On colposcopic examination uncertain--i.e. insignificant--results were recorded in 24.6%, on cytological examination in 19.7%. In patients where these methods gave unequivocal results (either + or-) a correct forecast of the presence of HPV during colposcopic examination was recorded in 71.1%, in cytological examinations in 66.9%. At least one of the clinical methods assessing papilloma virus infection was prognostically correct in 90.4%. From the investigation ensures that prebioptic methods provide the clinician with relatively reliable information on the presence of HPV infection and enable him to select a therapeutic and dispensarization procedure adequate to the finding. However, they cannot replace virological examination among other reasons also because they cannot assess the HPV type.

[Interleukin-6 and acute phase reactants in the diagnosis of ovarian carcinoma]. / Interleukin-6 a reaktanty akutní fáze v diagnostice ovariálního karcinomu.

In 115 women (healthy controls and patients with benign and malignant gynaecological tumors) interleukin-6 was determined in blood plasma with the aim to decide whether elevated IL-6 levels may be used as a marker of ovarian carcinoma. In spite of statistically significantly increased IL-6 levels the authors do not regard at present the IL-6 values as a useful marker of ovarian carcinoma for two reasons: first, until now it is not decided whether elevated IL-6 values originate only from the cells of epithelial ovarian carcinoma or if they are also produced by tumour-associated macrophages or both and second: in a large number of cases (both controls and patients with malignant tumors) no IL-6 levels in blood plasma could be detected. For these reasons it seems to be more convenient (even economically) to determine in suspected cases and after exclusion of any inflammatory process the levels of prealbumin and transferrin. Significantly decreased levels of both have a high value of primary sensitivity (66% and 87% resp.).

[Is it possible to predict the development of Nelson's syndrome in Cushing's disease after bilateral adrenalectomy?]. / Je mozná predikce vývoje Nelsonova syndromu u Cushingovy choroby po bilaterální adrenalektomii?

A detailed analysis of clinical and laboratory findings in a group of 66 patients with Cushing's disease treated with bilateral adrenalectomy, out of whom 15 developed Nelson's syndrome, gave evidence that Nelson's syndrome mostly affects children and young patients compared to older persons. Bilateral adrenalectomy performed in patients aged over 40 years was never accompanied by Nelson's syndrome. Basal plasma ACTH values prior to adrenalectomy (30.9 +/- 4.53 pmol/l in the absence of Nelson's syndrome and 31.3 +/- 5.41 pmol/l in patients with Nelson's syndrome) as well as the degree of their suppressibility with 8 mg dexamethane (to 18.8 +/- 3.43 pmol/l in Nelson's syndrome and 19.0 +/- 3.44 pmol/l in patients without it) did not provide sufficient evidence for the prediction of later development of Nelson's syndrome. The same is true of the plasma cortisol concentrations. A tendency to a significant plasma ACTH increase after adrenalectomy within a 6-month period, and especially its lesser suppressibility raises a strong suspicion of an incipient development of Nelson's syndrome. These findings will have to be taken into account when deciding on the surgical treatment of Cushing's disease.

[Papillomaviruses and human tumors]. / Vztah papillomaviru k lidským nádorum.

The report summarizes the main results obtained in the course of our research project. The results of immunological and epidemiological studies provide further proofs that human papillomaviruses (HPV) are the etiological agents in cervical neoplasia. In addition, they raise hopes that immunological methods may be utilized in diagnostics of cervical cancer and for monitoring the clinical course of this disease in the near future. Since the etiological relationship between HPV and cervical carcinoma seems to be proven beyond reasonable doubt, the development of prophylactic and therapeutic vaccines has become the dominant of the contemporary HPV reseach. For studying immune reactions against HPV-induced tumours we developed a model of HPV16-transformed rodent cells.

[Bilateral adrenocortical adenomas and the pheochromocytoma syndrome]. / Bilaterální adrenokortikální adenom se syndromem feochromocytomu.

A 43-year-old man with clinical signs of pheochromocytoma and adrenal adrenal enlargement underwent adrenalectomy. Bilateral adrenocortical adenomas accompanied by a pressure atrophy of surrounding cortex and normal adrenal medulla were found. The tumours exhibited neither immunohistochemical nor ultrastructural signs of medullar differentiation. As the clinical symptoms of pheochromocytoma slowly disappeared during the 18 month-postoperative period we consider the local tumour activity most probably responsible for the pseudopheochromocytoma symptomatology.