[Protein bcl-2 expression in the secretory endometrium after oral estrogen-progesterone replacement]. / Exprese proteinu bcl-2 v sekrecním endometriu po perorální estrogen-progesteronové substituci.

OBJECTIVE: To compare the changes of the protein bcl-2 expression, regulatory mechanism in the process of apoptosis, in the secretory endometrium throughout natural and artificial (estrogen-progesterone substitution) cycles in the same group of patients. DESIGN: A prospective study. SETTING: Dept. of Obstetrics and Gynecology, University Hospital Olomouc, Dept. of Histology and Embryology, Faculty of Medicine, Palacký University in Olomouc, Dept. of Obstetrics and Gynecology, Bata Hospital, Zlín. METHODS: Endometrial samples (n=56) were obtained on days 5 and 7 after ovulation, progesterone addition, resp. Patients (n=14) included in the study had regular menstrual cycle, age under 40, BMI range 20-32 and basal FSH level range 5-9 IU/l. The collected samples were processed routinely and bcl-2 was estimated by indirect three level imunohistochemic method. RESULTS: We demonstrated the bcl-2 expression in all evaluated layers (epithelial surface, stroma, glands) of the mid-secretory endometrium in both cycles. No difference was found in bcl-2 expression between days 5 and 7 in the secretory phase of the natural cycle. In artificial cycles higher bcl-2 expression was found only in epithelial surface (p<0.05) between days 5 and 7. On day 7 higher bcl-2 expression was found in the artificial cycle in the endometrial epithelial surface (p<0.001) as well as in spongy layer (p<0.01) and compact layer (p<0.05) comparing to day 7 of the natural cycle. CONCLUSION: The bcl-2 expression in the mid-secretory endometrium is significantly higher in the cycle with estrogen-progesterone substitution comparing to the natural cycle. These changes were more significant in endometrial and glandular epithelium than in stromal cells.

Changes in the development of uterine pinopodes in steroid hormone supplemented cycles.

The endometrium acquires the ability to implant a hatched blastocyst only within a specific time termed the receptive phase. Ovarian steroid hormones are essential for structural and functional changes that prepare the endometrium to be receptive. Pinopodes have been suggested to be markers of uterine receptivity. The aim of this study was to compare the pinopode expression and serum levels of ovarian steroid hormones in the mid-luteal phase of the natural cycle and in a "mock" cycle in the same subject. Sequentional endometrial biopsies within 48 hours were obtained from women in the mid-luteal phase (ovulation +5, ovulation +7) of the natural cycle and in the "mock" cycle (progesterone supplementation +5 and +7). Biopsies were examined under a scanning electron microscope for pinopode detection. The expression of pinopodes was similar in both cycles, where pinopodes covered about 5 % of the endometrial surface. The developmental stages were also similar with a slight increase of fully developed pinopodes in both samples in the "mock" cycles. Our findings suggest that hormonal preparation of the endometrium do not change the timing of pinopode expression.

Involvement of p53 and Bcl-2 family proteins in regulating programmed cell death and proliferation in human embryogenesis.

Homeostasis and development in vertebrates are regulated by cell proliferation, differentiation and death. Permeability of mitochondrial membranes, a decisive feature of apoptosis, is regulated by Bcl-2 family regulators. Protein p53 is able to reduce bcl-2 and promote bax expression. This study focused on the immunohistochemical detection of the expression levels of Bcl-2 family regulators (anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bcl-Xs and Bax), p53, and PCNA as a marker of proliferation, together with the evaluation of the level of apoptosis in human embryos (anlage of limbs, axial skeleton, metanephros, and intestine). Expression of observed proteins was assessed by a three-step immunohistochemistry and evidenced by the double-staining technique. Apoptosis was detected by the TUNEL technique. This study provided circumstantial evidence of the exclusive role of Bcl-2 and Bcl-XL proteins in the inhibition of apoptosis - only rarely were the Bcl-2/ Bcl-XL positive cells stained by TUNEL. The role of pro-apoptotic members of Bcl-2 family remains ambiguous, as TUNEL positive cells are both Bax/Bcl-Xs positive and negative. This study provided substantial evidence that expression patterns of observed proteins are neither fully explainable by "rheostat" theory, nor are the findings obtained from animal model tissue or cell culture commonly applicable to human embryos.

Apoptosis and expression of proliferative proteins in the developing nervous system and orofacial region of human embryos.

We have studied expression of PCNA and Ki-67 in the developing nervous system, sensory organs and orofacial regions in human embryos and fetuses, using monoclonal antibodies PC-10 and MIB-1 in three-step immunohistochemical method and apoptosis performed by TUNEL technique. Expression of PCNA and Ki-67 increased with the age. Apoptosis was rare in above mentioned regions.

The role of the apoptosis and the genes regulating apoptosis in the early differentiation of human embryo.

Apoptosis (programmed cell death) is an important process participating in the formation of organs and tissues during embryogenesis. Our aim of the work is studying the role of the apoptosis during the human embryonic differentiation. We tend to give acquired findings into the correlation with expression of proteins Bcl-2 and Bax (products of genes regulating apoptosis). Detection of the apoptosis was carried out on 25 routinely processed human embryos by means of TUNEL technique. The level of expression of Bcl-2 and Bax was determined using standard three-step immunohistochemical procedure. Results were achieved by the comparison of apoptoic index and the level expression of Bcl-2 and Bax was semiquantitatively evaluated. The low value of apoptotic index was mostly accompanied by the high expression of Bcl-2 and the Bax expression was not proportionally related to the value of apoptic index.

[Comparison of apoptosis expression in the secretory endometrium during a normal menstrual cycle]. / Porovnání exprese apoptózy v sekrecním endometriu prirozeného menstruacního cyklu.

OBJECTIVE: Comparison of level of apoptosis in the mid-secretory human endometrium. DESIGN: Clinical-laboratory trial. SETTING: Department of Gynecology and Obstetrics, Medical Faculty, Palacký University, Faculty Hospital, Olomouc, Department of Gynecology and Obstetrics, Bata Hospital, Zlín. METHODS: Samples of secretory endometrium were obtained from 14 women. The women included in the study had the next criteria: a history of infertility of more than 12 months, regular menstrual cycles, age below 40 and FSH basal level range 5-9 IU/L. Sampling of tissue was performed after confirmed ovulation by LH surge and repeated ultrasound examinations. Patterns were processed by routine methods and apoptosis was detected using TUNEL assay. RESULTS: The level of apoptosis in the endometrial epithelial surface was significantly higher on day +7 compared to day after ovulation (P < 0.02). Apoptotic cells were seen only sporadically in endometrial stromal cells. The level of apoptosis in endometrial stroma was also higher (but not statistically significant) on day +7 compared to day The level of apoptosis was markedly higher in spongy layer on day +7 (P < 0.01) compared to day There were no significant differences between day and +7 in compact layers of the glands.

[Disorders of the heart conduction system]. / Nemoc prevodního systému srdecního.

Disorders of the genesis and conduction of impulses in different parts of the cardiac conduction system are the result of functional or morphological damage of parts of the conduction system. Pathological processes which affect the cardiac conduction system are practically always comprehensive and ensue from the character of the basic disease. Previous embryological and histological investigations provide evidence of developmental and structural similarity of all portions of the cardiac conduction system. On this fact the assumption of a similar reaction of the conduction system to the acting pathological process is based. In a group of 127 patients with a positive finding, on invasive examination of the conduction system in 57 (45%) a pathological process was detected at several levels of the conduction system. This finding is consistent with experience reported in the literature and supports the justification of the term "disease of the cardiac conduction system" when there is evidence of multi-focal damage of the conduction system.

Endometrial expression of the estrogen-sensitive genes MMP-26 and TIMP-4 is altered by a substitution protocol without down-regulation in IVF patients.

BACKGROUND: The aim of this study was to analyse the effects of an estradiol (E(2))-progesterone substitution protocol on the endometrial expression of estrogen-sensitive genes during the peri-implantation period. METHODS: Peripheral blood and endometrial biopsies were obtained from 13 infertile women both in a natural cycle (NC), on days 5 and 7 after ovulation (NC5, NC7), and in an artificial (substituted) cycle (AC), on days 5 and 7 of progesterone addition (AC5, AC7). Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were assayed by immunohistochemistry. Matrix metalloproteinase-26 (MMP-26) mRNA and tissue inhibitor of metalloproteinase-4 (TIMP-4) mRNA were semiquantitatively assessed in tissue sections using in situ hybridization (ISH) and quantified in tissue extracts using real-time PCR. RESULTS: Levels of both E(2) and progesterone were higher in the peripheral blood in AC than in NC. Also on day AC5, expressions of ERalpha, PR and MMP-26 mRNA (focally) were increased in the epithelium and TIMP-4 mRNA in the stroma. Expression levels of these genes dropped significantly between AC5 and AC7, but not between NC5 and NC7. Abnormally high levels in AC5 samples suggest overstimulation with E(2), and the rapid decrease between AC5 and AC7 suggests overstimulation with progesterone. CONCLUSIONS: In ACs, increased levels of E(2) in the blood exaggerate the endometrial expression of estrogen-sensitive genes, whereas higher levels of progesterone in the blood in the secretory phase exaggerate the drop in expression of these genes. Dramatic variations in the gene expression may not be optimal for the implantation process.

Enzyme histochemistry in the developing suprarenal gland of human embryos.

The localization of the activities of some selected phosphatases, peptidases and dehydrogenases were studied in cryostat sections of the developing anlage of the suprarenal gland of human embryos from 8 to 20 weeks of the intra-uterine life. In the youngest fetuses under our notice (weeks 8-12), the activity of alkaline phosphatase (AP) on the cellular membranes of the fetal cortex was very low. In contrast, the activity of acid phosphatase (AcP) was comparatively high. Peak activity was found in the cells of the central zone of the fetal cortex. Compared to the activity of the latter, the activity of non-specific esterase (ANE) was somewhat lower. Both its localization and the gradient were identical with those of acid phosphatase. Of the peptidases studied, only dipeptidyl peptidase IV (DPP IV) exhibited slight activity in deeper layers of the primitive fetal cortex after week 8. The other peptidases exhibited only traces of activity. As early as in the first stages followed, the activity of glycero-3-phosphate-dehydrogenase (alpha-GPDH) was very high in all cells of the differentiating fetal cortex. The intensity of the activity of succinate dehydrogenase (SDH) was markedly lower. In older fetuses (weeks 13-20) there was a gradual increase in the activities of most enzymes, seen, after week 15 of the intrauterine life, also in the cells of the so-called definitive cortex. Most pronounced were the increases in the activities of acid phosphatase and non-specific esterase. The relatively low activities of the enzymes under study point to a relatively low degree of cell differentiation of both the primitive and, after week 15, the definitive cortex. Pronounced morphological and functional changes occur after the 20th week of the intrauterine life.

Enzyme histochemistry in fetal human hearts and kidneys during intrauterine development. Qualitative and quantitative results.

The activities of three enzymes, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and dipeptidylpeptidase IV, were histochemically demonstrated and cytophotometrically measured in fetal human hearts and kidneys. During the intrauterine development from the 7th to the 19th week 29 fetuses in 6 stages of age were investigated. Positive enzyme reaction was found in the myocardium of left and right ventricle and septum and in the different parts of the nephron. During the investigated period of development the enzyme activities changed in both heart and kidney. At the 16th week the activities of glycerol-3-phosphate dehydrogenase and succinate dehydrogenase showed the maximum in the heart, in contrast to kidney, where at this stage activity minima of succinate dehydrogenase and dipeptidylpeptidase IV were measured. The myocardium of the right ventricle showed higher metabolic activity than the right one, which could reflect the higher load of the right ventricle in the fetal circulation. Within the nephron differences of enzyme activities were observed. The highest enzyme activities were always measured in the proximal tubules, interpreted as sign of high degree of differentiation.

[Differentiation of the human heart. II. Review of histochemical differentiation]. / Die Differenzierung des menschlichen Herzens. II. Die Ubersicht der histochemischen Differenzierung.

Histochemical differentiation of the myocardium is characterized by a gradual increase in the number of demonstrated enzymes and by their enhanced activity. The enzymatic heterogeneity of the endothelium of myocardial capillaries can be proved from the beginning of the fetal development. All findings were obtained from the study of human material.

Multifocal disturbance of cardiac conduction system.

The group of 127 patients with pathological finding in cardiac conduction system revealed by electrophysiological invasive examination was evaluated with regard to localization of conduction disturbance. A single focus was observed in 70 patients, multifocal affection was found in 57 cases (45%). This finding is in agreement with literary data and thus the use of the term "disease of cardiac conduction system" is recommended in all cases where multifocal disturbance of conduction system is diagnosed.

[Morphologic changes in lung tissue in mice after long-term exposure to NO2]. / Die morphologische Veränderungen des Lungengewebes der Mäuse nach der langfristigen Exposition mit NO2.

The light microscopical, histochemical and ultrastructural changes induced by NO2 were studied in lung tissue of experimental mice. The animals were daily exposed to NO2 for one hour and after 12 days we observed focal light microscopical changes (dilatation of alveolar crypts, minute inflammatory infiltrates). A rather high point activity of acid phosphatase was found, particularly in the cytoplasm of alveolar and septal macrophages. The light microscopical findings were supported by ultrastructural changes, i.e. vacuolation up to disintegration of alveolar pneumocytes, multiplication of macrophages, thickening of alveolar septa with multiplication of collagenous fibrils and infiltrate consisting of lymphocytes and plasma cells. The endothelium of capillaries displayed vacuolation and thickening of basal membrane in some sections. The quantitative examination showed multiplication of goblet cells in the epithelium of bronchial stroma of experimental animals compared to the controls. No morphological changes in the myocardium of the control group were observed even at ultrastructural level.

Histochemistry of enzymes in the pancreas of human embryos.

12 human embryos and fetuses (in weeks 4 to 20 of the intrauterine life) were studied using the methods according to Lojda for the activity of the following enzymes: alkaline and acid phosphatases (AlP, AcP), acid nonspecific esterase (AE), ATP- splitting enzymes (ATP-ase), beta-glucuronidase, aminopeptidases A and M (APA, APM), dipeptidylpeptidase IV (DPP IV), gamma-glutamyltransferase (GGT), succinate dehydrogenase (SDH), and glycero-3-phosphate dehydrogenase (alpha-GPDH). Glycogen content was determined by PAS method. In the youngest embryos, a high activity of DPP IV was recorded in the epithelium of differentiating primitive glandular tubules. Activity of other peptidases was low. The activity of AcP was found in tubular epithelium and mesenchymal cells. After week 7, glycogen was present in the supranuclear zone of tubule epithelium. In older fetuses, especially after week 15 of the intrauterine life, the activity of all studied enzymes gradually intensifies. In acinic anlage, a high activity of DPP IV was observed, activity of APM and GGT increased, activity of APA was lower. A relatively high activity of peptidases was recorded even in the epithelium of ducts. The capillaries showed a high activity of AlP and ATP-ase.

Enzymatic equipment of the nasal mucosa in the developing human nasal cavity.

The activities of some enzymes were described in the developing human nasal cavity in relatively early stages. In cryostat sections of the 8-14-week-old fetuses processed by standard Lojda methods, activities of the following enzymes were found in the respiratory and olfactory regions: ALP, ACP, ATP, TPP, alpha-GPDH, G6PDH, SDH and ANE. The positive reaction for DPP IV failed to be proved in this localization, it was not detected even in the capillary bed of the nasal cavity.

Quantitative histochemical study of differentiation of bile canaliculi and liver sinusoids in the liver of human embryos.

The study was performed on the liver tissue of 15 human embryos and foetuses aged 7 to 19 weeks. The combined reaction to dipeptidylpeptidase IV (DPP IV) and alkaline phosphatase (AlP) was performed on cryostat sections. A test grid according to Weibel was used for the evaluation of the DPP IV and AlP-positive areas. During the whole period studied the presence of AlP positive areas prevailed over that of DPP IV positive ones, while the number of areas without activity remained unchanged. The AlP-positive areas vary between 15-29%, the extent of DPP IV positive areas is in the range of 11-18%.

Early prenatal development of the brush border enzymes in the embryonal intestine.

Standard methods were used for the study of the activity of brush border enzymes in the epithelium of the intestine of 45 human embryos aged from 4 to 22 weeks of the intrauterine life. Enzymes of the peptidase group (DPP IV, APM, APA, GGT, BBEP), of the phosphatase group (ALP, ACP, AMP, ATP), and of the disaccharidase group (saccharose, lactase, trehalase) were studied. In the embryonal developmental period (to week 8), the activity of proteases was mainly detected on the luminal surface of the primitive pseudostratified columnar epithelium of the intestine anlage. The highest activity was displayed by DPP IV. In the period from the 8th week, villi are formed from the duodenum up to the ileum. After week 9, differentiation of Lieberkühn crypts was observed. The activity of proteases was very high (DPP IV especially) in the differentiating microvillous zone of primitive enterocytes. Gradient of apex-base activity of the villus was maximal on the apex of villi. After week 12 of the intrauterine life, the activity of disaccharidases ALP and AMP occurs in the differentiating brush border, and a low activity of BBEP of the protease group is present.

[Distribution of the activity of some peptidases in the pancreas of human embryo]. / Distribution der Aktivität einiger Peptidasen im Pankreas der menschlichen Keimlinge.

In the developing pancreas of human embryos and fetusses (in weeks 4 to 20 of the intrauterine life) activities of aminopeptidase A (APA) and M (APM), dipeptidyl peptidase IV (DPP IV), and gamma-glutamyl transferase (GGT) were studied in cryostat sections using methods as recommended by Lojda (J. Histochem. Cytochem. 29, 481 (1981)). In the youngest embryos (4-7 weeks) a high DPP IV activity was recorded in the epithelium of differentiating primitive glandular tubules. Activity of other peptidase was low. In older fetusses (8-20 weeks, particularly after week 15) activities of all peptidases studied gradually increase. In acinic "anlage" a high DPP IV activity was observed. APM activity was substantially lower. The reaction product originating in the reaction for GGT is localized particularly at the luminal surfase of epithelial lining of primitive tubuli. In this period APA activity was hardly discernible. This is in contrast with other organs. High DPP IV and APM activities were present in cells of differentiating Langerhans islets. The appearance of peptidases studied does not occur simultaneously, however, their activities increase with the morphological differentiation.

[Histochemical analysis of enzymes in the rabbit cornea after LASIK surgery]. / Histochemické vysetrení aktivity nekterých enzymu v králicí rohovce po operaci LASIK.

The first experience with histochemistry of rabbit corneas after the operation LASIK is presented in this study. Activity of the following enzymes was assessed: alkalic phosphatase, acid phosphatase and dipeptidylpeptidase IV. No pathological activity of assessed enzymes was found in comparison with control corneas.