Effects of vinclozolin exposure on the expression and activity of SIRT1 and SIRT6 in the porcine ovary.

Within the mammalian reproductive system sirtuin 1 and 6 (SIRT1, SIRT6) are considered to contribute to steroid hormone signaling and control of reproductive physiology. Therefore, the specific question is whether and how a commonly used dicarboximide fungicide with antiandrogenic activity, vinclozolin (Vnz) alters SIRT1 and SIRT6 expression and whether both investigated sirtuins positively affect survival of the follicles after vinclozolin exposure. Immunocytochemistry and immunohistochemistry were performed to localize SIRT1 and SIRT6 expression in cultured granulosa cells (GCs; 48 hours) and whole ovarian follicles (24 hours) after treatment with two androgens, testosterone (T; 10-7 M) and dihydrotestosterone (DHT; 10-7 M), and an antiandrogen, Vnz (1.4 x 10-5 M), separately and in combinations. Granulosal and follicular mRNA and protein expression of both sirtuins was also investigated by real-time PCR and Western blot. In addition, their concentration and activity was studied by immunoenzymatic and fluorescence assays. Our observations: (1) demonstrate the presence of both investigated sirtuins in ovarian cells, (2) show their potential involvement in the control of follicular atresia because of increased SIRT1/SIRT6 expression and SIRT1 activity after exposure to Vnz, (3) represent the first data on the interrelationships between sirtuins and androgens in porcine ovarian cells. Based on these findings and our previous results we can conclude, that SIRT1 and SIRT6 do not exert the protective effects in ovarian follicles after vinclozolin exposure. These novel data on the role of SIRT1/SIRT6 in porcine ovarian follicles shows that in the presence of the investigated fungicide, sirtuins are upregulated, which can induce apoptosis of follicular cells. Furthermore the androgen receptor sensitivity to ligands, especially environmental ones (for example: vinclozolin) might be directly linked with the mechanism of action of both investigated sirtuins in the porcine ovary, which requires further investigation.

The influence of the antiandrogen 2-hydroxyflutamide on the androgen receptor expression in the porcine ovarian follicles - an in vitro study.

Androgens are one of the most important agents influencing ovarian follicles growth and development. The biological action of androgens is primarily exerted through transcriptional regulation by the androgen receptor (AR), a member of the steroid hormone receptor superfamily. The purpose of this study was to test the role of androgen receptor agonist testosterone (T) or antagonist 2-hydroxyflutamide (2-Hf) and in combination on AR expression in cultured porcine granulosa cells (GC) or whole follicles. Granulosa cells isolated from mature pig follicles were cultured for 48 h. During the last 12 and 24 h of culture, they were incubated in the presence of T (10(-7)  m/ml), 2-Hf (1.7 × 10(-4)  m) or both T and 2-Hf (T + 2-Hf, at the same concentrations as when added separately). To better imitate in vivo conditions, whole follicles (6-8 mm in diameter) isolated from porcine ovaries have been incubated (for 12 and 24 h) in an organ culture system with the addition of the same factors. Thereafter, cells or sections obtained from cultured follicles were processed for AR detection by immunocytochemistry or immunohistochemistry. Moreover, expression of AR mRNA and protein was determined by real-time PCR and Western blot analysis. It was shown that the addition of 2-Hf in the presence of T had a positive effect on AR mRNA and protein expression in porcine GC and ovarian follicles. Moreover, the addition of 2-Hf influenced AR distribution in GC cultures which is seen as change of its localization from nuclear to perinuclear. Our results suggest that androgens acting through AR could be involved in the control of AR expression in porcine GC in vitro and in vivo.

Haematopoiesis in snakes (Ophidia) in early postnatal development.

The occurrence of haematopoiesis has been studied in various parts of the spine and in the ribs in four species of snakes (Boa constrictor L., Elaphe guttata L., Lamprophis fulaginosus Boie., Bothrops jararaca Wied.) from hatching until 150 days of postnatal development. Marrow spaces are formed by chondrolysis with various time frames depending on the studied species. Marrow cells egress to the general circulation in two ways: via migration through the endothelial cells lining the venous sinuses or by the rupture of protrusions. Erythroblasts are present in the lumen of marrow sinuses suggesting their final maturation there. Various relationships of the spleen to the pancreas have been found. No myelopoietic foci occur in the spleen, liver or kidney of any of the studied species. However, erythropoiesis (sparse islets) has been observed in Bothrops jararaca spleen.

Complementary approaches demonstrate that cellular aromatization in the bank vole testis is related to photoperiod.

A growing body of evidence indicates that germ cells, at least in several mammalian species, are responsible for estrogen formation since they possess active aromatase. In seasonally breeding rodent, the bank vole, the length of photoperiod seems to be the primary environmental factor regulating annual changes in the reproductive activity. However, in this species gonadal steroidogenesis is still not well understood, neither the site of aromatization in testicular cells. In the bank vole testis, aromatase visualized by immunohistochemistry was found in Leydig cells, Sertoli cells, and germ cells: especially in spermatocytes and spermatids. Moreover, in the immuno-electron microscopic study, gold particles indicating aromatase were observed over the cytoplasm of elongated spermatids. The presence of aromatase and the activity of this enzyme were found in microsomal preparations of the whole testes and those of seminiferous tubules. This was measured by means of Western blot and the biochemical assay with tritiated androstenedione, respectively. Additionally, using radioimmunological assays testosterone and estradiol concentrations in homogenates were detected. All the studied parameters revealed close correlation with the length of photoperiod being evidently higher in animals kept in the long day conditions when compared with those from short light cycles.

Haematopoiesis and a new mechanism for the release of mature blood cells from the bone marrow into the circulation in snakes (Ophidia).

This is the first description of haematopoiesis in snakes. Studies were carried out on the following species belonging to Ophidia: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe taeniura taeniura, Boa constrictor,and Python reticulatus. Smears of the peripheral blood and histological preparations from the vertebrae, ribs, liver, and spleen were studied under a light and electron microscope. Myeloid cells were present in the following locations in the vertebrae: the neural spine, zygoapophysial processes, floor of the neural canal, lacunae in the bodies of vertebrae and also inside the ribs. Although the vascular system was well developed, especially around the ribs, vessels inside the marrow cavities were scarce, both in the ribs and elsewhere where haematopoiesis was found. Venous sinuses were well developed in the vertebrae and in the rib regions from their costal head towards the middle area. They consisted of one layer of fine endothelial cells. Mature cells in the process of migration into the general circulation were only sporadically encountered when venous sinuses were studied on perfusion-fixed specimens. In contrast, almost every sinus venosus contained protrusions directed towards the lumen, filled mostly with mature and immature blood cells. Various stages of their formation were seen in the cross sections of venous sinuses ranging from small, newly formed to large, elongated ones, filled with many fully developed and some maturing blood cells. In many cases the apices of the protrusions were ruptured, and mature blood cells, as well as a few immature ones, were seen in their vicinity. This observation led us to a new hypothesis that blood cells are released from the extravascular space into the lumen of venous sinuses. In snakes, these cells are released into the systemic circulation mainly via the rupture of protrusions filled with mature blood cells and, to a lesser degree, by transcytosis as known in mammals. In the spleens from young specimens, 1-2 foci of haematopoiesis were encountered where lymphopoiesis predominated. Haematopoiesis was not detected in the liver.

Hematopoiesis in snakes (Ophidia).

Locations of the hematopoietic tissue have been described in the following ophidian species: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe teniura teniura, Boa constrictor, and Python reticulatus. Studies were carried out on perfusion fixed vertebrae, ribs, spleen, liver, thymus, and kidney. Routine histological technique was applied using both light and electron microscopy. Hematopoietic tissue was found in the following locations of the vertebrae: neural spine, neural arch, postzygophysis processes, hypapophysis, vertebral centre. Moreover, intense hematopoiesis was found inside the ribs. In the spleen and thymus, only lymphopoiesis was found. Hematopoietic islets in the spleen were sporadically found only in young specimens. No hematopoiesis was observed in the liver and kidney. In the studied species, there were no differences in the location of hematopoietic tissue. A new model of mature and immature blood cell release to the lumen of marrow sinuses different from that known to operate in higher vertebrates is proposed.

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary.

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.

Immunological findings in cigarette smokers.

The aim of our study was to assess the effects of cigarette smoking on selected indices of immunity. The study comprised 116 men divided into three groups: 37 subjects smoking for not more than 10 yr, 39 subjects smoking for more than 10 yr, and control group consisting of 40 age-matched men who never used to smoke. The following parameters were studied: total number of lymphocytes, B-cells, T-cells subpopulations: (CD3+)T-, (CD4+)T-helper, (CD8+)T-cytotoxic and (CD16+)natural killer (NK)-cells and serum concentration of immunoglobulins A, D, G and M, C3c and C4 complement components, acute phase proteins: alpha(1)-acid glycoprotein, haptoglobin, ceruloplasmin and lysozyme. The (CD4+)/(CD8+) ratio was also calculated. The suppressive effect of tobacco smoke on human immunity was seen as decreased serum concentration of immunoglobulins and lysozyme, especially in men smoking for more than 10 yr, decreased (CD16+)NK-cells absolute number and elevated population of (CD8+)T-cytotoxic lymphocytes entailing a decrease in CD4+/CD8+ ratio.

Apoptosis of postovulatory cumulus granulosa cells of the rat.

The process of apoptosis in the postovulatory cumulus granulosa cells was investigated in pregnant rats. Mature female Wistar rats, exhibiting a regular 4-day oestrous cycle, were placed with males on the day of pro-oestrus. The following day, on which spermatozoa were found in vaginal smears, was designated day 1 of pregnancy. The animals were killed just before ovulation (24.00 hours), on days 1 (5.00, 11.00, and 18.00 hours), and 2 ( 11.00 hours) of pregnancy. Excised ovaries and oviducts were submitted to a routine histological procedure and paraplast sections were subjected to detection of apoptotic cells using the TUNEL method. The cumulus granulosa cells of preovulatory follicles (24.00 hours) were negative for apoptotic staining. However, 5 h later a positive staining was observed in the oviduct ampulla and included the cumulus granulosa cells lying in the peripheral parts of postovulatory cumuli oophori, and the oviductal epithelial cells of this region. On the evening of day 1 almost all cumulus granulosa cells showed strong immunostaining while on day 2 at 11.00 hours only immunonegative clusters of remnants of cumulus granulosa cells were present in the distended ampulla region, while naked, two or more cell embryos were present in the further parts of oviduct. These results indicate that in the rat apoptosis of cumulus granulosa cells starts shortly after ovulation in the peripheral region. Epithelial ampullary cells surrounding ovulated cumuli show a massive apoptosis.

Noradrenergic and peptidergic innervation of the mouse femur bone marrow.

The innervation of bone marrow from femur bones of BALB/c mice was studied by means of immunohistochemistry and fluorescence histochemistry. The immunoperoxidase method with nickel amplification was applied to visualize the topographical distribution of nerve fibers using antibodies against the general neuronal marker PGP 9.5 (neuron-specific cytoplasmic protein), catecholamine synthesizing enzyme tyrosine hydroxylase (TH) and neuropeptide Y (NPY). Glyoxylic acid-induced fluorescence was also applied to demonstrate catecholamine-containing nerves. Both staining methods revealed dense innervation by fibers seen predominantly around blood vessels but also ramifying among marrow cells. Recent findings on adrenergic and peptidergic influences on marrow physiology combined with anatomical data indicate the existence of a neural modulation of hematopoiesis.

Some morphological and hormonal aspects of ovulation and superovulation in the rat.

In the first experiment, mature female Wistar rats, displaying a regular 4-day oestrous cycle, were killed in succession every 2 or 3 h on the day of pro-oestrus and oestrus until the time of ovulation. In the second experiment, immature female Wistar rats (aged 24 days) were injected s.c. with 30 IU pregnant mare serum gonadotrophin (PMSG) and 56 h later with 20 IU human chorionic gonadotrophin (hCG). They were killed in groups at 0, 24, 48, 56 and 57 h, and then every 2 h until 72 h. Excised ovaries were homogenized and analysed for steroid content or they were submitted to a routine histological procedure. The cyclic and PMSG/hCG-treated rats exhibited some similarities and differences in the general pattern of steroid content. Either a presumptive endogenous LH surge or administration of hCG resulted in an increase in the ovarian androgen concentration which preceded a rise in progesterone; the progesterone peak, in turn, was accompanied by a fall in the amount of androgens and oestradiol. However, in comparison with cyclic rats, superovulated animals displayed a significantly higher ovarian androgen level for a prolonged period; ovarian oestradiol concentration was also raised while the progesterone content was much lower. Histological analysis revealed large differences between the ovaries of superovulated and cyclic rats, especially with regard to the maturing follicles. The majority of PMSG/hCG-derived follicles showed hypertrophied theca interna and degenerated or luteinized granulosa. A large number of preovulatory follicles did not ovulate. These results clearly indicate that PMSG/hCG-induced follicles are not equal to the follicles developing during a normal oestrous cycle. This should be taken into consideration when using superovulated animals in experiments.

Effect of small doses of ethylene glycol monomethyl ether on the acetylcholinesterase and delta-aminolevulinic acid dehydratase activity in erythrocytes, blood and bone marrow of rats.

The effect of small doses of ethylene glycol monomethyl ether (EGMME) on the activity of acetylcholinesterase (ACHE) in erythrocytes and whole blood as well as on the delta-aminolevulinic acid dehydratase (ALA-D) in blood and bone marrow was studied in Wistar rats. Significant reduction in the activity of both enzymes was noted three days after ip administration of 200 mg/kg b.w. EGMME whereas seven days later the activity of both enzymes returned to the control levels. Activity of ALA-D in blood appeared to be most sensitive to EGMME, and reacted even to the lowest dose, which did not significantly alter activity of ACHE or ALA-D in bone marrow. Haematological parameters in all treated groups remained unaltered.

Effect of haemodialysate and its peptide fractions on acetylcholinesterase activity in erythrocytes from healthy subjects and patients with terminal renal failure.

The effects of haemodialysate and its three peptide fractions on acetylcholinesterase (AChE, E.C. 3.1.1.7) activity in erythrocytes from healthy subjects and patients with terminal renal insufficiency treated by repeated haemodialyses have been studied. It was shown that erythrocytes from haemodialysed patients display an increased activity of the enzyme if compared with those from healthy subjects. Neither haemodialysate nor any of its three peptide fractions, when incubated with erythrocytes from healthy subjects and from haemodialysed patients, have altered the activity of the enzyme, except for fraction III at the highest concentration. This fraction exerted an inhibitory effect on AChE activity of the erythrocyte derived from healthy subjects.

[Activity of selected enzymes of peripheral blood erythrocytes and serum haptoglobin levels in workers occupationally exposed to styrene and formaldehyde]. / Aktywnosc wybranych enzymów erytrocytów krwi obwodowej oraz poziomy haptoglobin w surowicy krwi pracowników eksponowanych na dzialanie styrenu i formaldehydu.

In 48 and 30 workers exposed to styrene and formaldehyde respectively activities of erythrocyte acetylcholinesterase lactate dehydrogenase and glucose-6-phosphate dehydrogenase, were determined. Hematocrit, haemoglobin concentration, red blood cell count and serum haptoglobin levels were also determined. Significant decrease in erythrocyte acetylcholinesterase activity in workers exposed to styrene for 61-180 months was stated. Moreover, increased erythrocyte lactate dehydrogenase activity and decreased serum haptoglobin level was found in workers exposed to formaldehyde for 3-24 months. There were no differences in basic hematological parameters and erythrocyte glucose-6-phosphate dehydrogenase activity in both groups studied as compared to the control group.

Effect of haemodialysate and its peptide fractions on lactate dehydrogenase activity in erythrocytes from healthy subjects and patients with end-stage renal disease.

The effect of haemodialysate and its 3 peptide fractions on lactate dehydrogenase activity in erythrocytes was studied in patients treated by repeated haemodialysis for end-stage renal disease and in healthy subjects. Erythrocytes from dialyzed patients showed significantly lower LDH activity than those from healthy subjects. After a 3-h incubation with haemodialysate (675 and 450 micrograms protein/ml), or its peptide fraction III (270 and 190 micrograms protein/ml), a significant inhibition of LDH activity was observed. On the other hand, neither haemodialysate nor its peptide fractions inhibited LDH activity in red blood cells from patients with end-stage renal disease treated by repeated haemodialysis.

[Changes in erythrocyte shape in children treated with chlorpromazine]. / Zmiany ksztaltu erytrocytów u dzieci leczonych chloropromazyna.

Children suffering from schizophrenic psychosis were treated with chlorpromazine for 21 days. Changes in erythrocyte shape were studied by scanning electron microscopy. It was found that the percentage of stomatocytes in sick children was significantly greater than in healthy children. There was no such a difference in case of echinocytes.

Histochemical localization of delta 5-3 beta-HSDH activity in preovulatory rat follicles.

Rat ovarian follicles were isolated at 3 different hours before ovulation (at 16, 24 and 2 h) and the activity of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) was examined by means of a new embedding technique. The activity of the enzyme in the theca interna cells, though the most intense one, was constant throughout the period of the time examined. It was the granulosa cells that showed the changing pattern of the activity: the reaction was strong there at 16 h and much weaker at 24 and 2 h (i.e. just before ovulation). The granulosa cells lying near the antrum displayed usually weaker reaction than those in the mural region. These differences in the enzyme activity between the mural and antral regions correlated with the morphological differenciation of granulosa cells into the two regions.