Morphological and functional adaptations of Fusobacterium nucleatum exposed to human neutrophil Peptide-1.

BACKGROUND AND OBJECTIVE: We recently demonstrated that Fusobacterium nucleatum can resist to human neutrophil peptide (HNP)-1 by decreasing its membrane permeability and increasing its proliferation and biofilm formation. In this continuation study, we aimed to further evaluate and explain these resistance properties by determining the morphological and functional adaptations of F. nucleatum, using transmission electron microscopy (TEM). MATERIALS AND METHODS: Cultures of the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were incubated without (0 µg/ml) or with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml). Membrane morphology and thickness, and cell (visualized by TEM), planktonic growth (measured in colony forming units), and biofilm formation (measured as total mass) were analyzed. Scrambled HNP-1 was used in planktonic growth and biofilm formation studies as a negative control. RESULTS: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. Intracellular granules became increasingly visible in the strain ATCC 25586 with increasing peptide concentrations. With increased concentrations of HNP-1, planktonic growth of the two clinical strains was significantly enhanced (P < 0.001) and of the type strain significantly suppressed (P < 0.01). HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 (P < 0.01) and 9508 (P < 0.001) significantly. Scrambled HNP-1 showed no effect on planktonic growth or biofilm formation of the tested strains. DISCUSSION: F. nucleatum has the ability to withstand the lethal effects of HNP-1, and the ultrastructural changes on bacterial membrane and cytoplasm may play role in this adaptive process.

Morphological abnormalities in gonads of the Baltic herring (Clupea harengus membras): Description of types and prevalence in the northern Baltic Sea.

Due to heavy anthropogenic influence and variation of the environmental conditions in the Baltic Sea, reproductive disorders are becoming a major environmental concern. We show here an increasing prevalence of gonadal malformations in the Baltic herring (Clupea harengus membras), a key species of the Baltic ecosystem and important in commercial fishery. During 1987-2014, the spawning herring population in the Archipelago Sea (AS) (North Baltic Sea, Finland) was monitored annually and analyzed for gross morphology of the gonads [total number (n) of analyzed fish = 38 284]. Four different types of malformations were repeatedly found and named as asymmetric, rudimentary, segmented, and branched gonads, but also hermaphroditic gonads and miscellaneous (unidentified) disorders were recorded. In 2013, additional samplings (n of fish analyzed = 541) showed similar malformations in herring from the Bothnian Sea. In some gonad types, histological examination revealed disintegration of seminiferous tubules and hyperplasia of the interstitial tissue. In 2014, the overall prevalence of malformations was still relatively low in the AS (frequency = 0-3.4 %; n = 750) and had apparently minimal effect on population recruitment. However, an increasing trend in the time-series (GLM; F = 32.65; p < 0.001) and a significantly higher prevalence in the Bothnian Sea (frequency = 0.7-5.0 %; n = 541; χ (2) = 6.24; p < 0.05) suggest that gonadal malformations may become a new threat for fish in the Baltic Sea. The observed gonad atrophies may be due to environmental endocrine disruption; however, also other explanations may exist and potential explanations are discussed.

Histological knowledge as a predictor of medical students' performance in diagnostic pathology.

Over the years, the role and extent of the basic sciences in medical curricula have been challenged by research on clinical expertise, clinical teachers, and medical students, as well as by the development and diversification of the medical curricula themselves. The aim of this study was to examine how prior knowledge of basic histology and histopathology among students predicts early learning of diagnostic pathology. Participants (N=118, representing 91% of the full student cohort) were medical students at the University of Turku, Finland. Data were collected during two preclinical courses that students attended in their first and second years of medical school. The measurements included tests on biomedical and clinical knowledge and a performance test in diagnostic pathology. Second-year performance on the diagnostic pathology examinations was predicted by the students' prior knowledge of histology, but not by the students' prior knowledge of histopathology. Although earlier research has demonstrated similar results in studies with shorter longitudinal designs, the present study demonstrates that the effect remains even if there is a considerably long time delay (a year) between the measurements, thus confirming the long-term value of basic science studies in the preclinical phase.

Antiandrogen exposure in utero disrupts expression of desert hedgehog and insulin-like factor 3 in the developing fetal rat testis.

Testicular development is an androgen-dependent process, and fetal exposure to antiandrogens disrupts male sexual differentiation. A variety of testicular disorders may result from impaired development of fetal Leydig and Sertoli cells. We hypothesized that antiandrogenic exposure during fetal development interferes with desert hedgehog (Dhh) signaling in the testis and results in impaired Leydig cell differentiation. Fetal rats were exposed in utero to the antiandrogen flutamide from 10.5 d post conception (dpc) until they were killed or delivery. Fetal testes were isolated at different time points during gestation and gene expression levels of Dhh, patched-1 (Ptc1), steroidogenic factor 1 (Sf1), cytochrome P450 side-chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1), and insulin-like factor 3 (Insl3) were analyzed. To study direct effects of hedgehog signaling on testicular development, testes from 14.5 dpc fetuses were cultured for 3 d in the presence of cyclopamine, sonic hedgehog, or vehicle, and gene expression levels and testosterone secretion were analyzed. Organ cultures were also analyzed histologically, and cleaved-caspase 3 immunohistochemistry was performed to assess apoptosis. In utero exposure to flutamide decreased expression levels of Dhh, Ptc1, Sf1, P450scc, Hsd3b1, and Insl3, particularly from 17.5 dpc onward. Inhibition of hedgehog signaling in testis cultures resulted in similar effects on gene expression levels. Apoptosis in Wolffian ducts was increased by cyclopamine compared with sonic hedgehog- or vehicle-treated cultures. We conclude that exposure to the antiandrogen flutamide interferes with Dhh signaling resulting in an impaired differentiation of the fetal Leydig cells and subsequently leading to abnormal testicular development and sexual differentiation.

Effects of the wood extractives dehydroabietic acid and betulinol on reproductive physiology of zebrafish (Danio rerio)-a two-generation study.

Two wood extractives, dehydroabietic acid (DHAA) and betulinol (BET), present in wood industry effluents were evaluated for their potential effects on the reproductive physiology of zebrafish. Adult zebrafish (F0) were exposed in a continuous flow-through system to 50 microg/l DHAA, 5 microg/l BET and 0.27 microg/l (1 nM) 17beta-estradiol (E2) for 3 months. Eggs were collected from F0 fish and the following F1 generation was exposed for 6 months. Biomarkers analyzed in both F0 and F1 fish were plasma vitellogenin (Vtg), testosterone (T), E2 (only females) and gonadal histology. DHAA and BET affected growth in terms of increased condition factor, and spawning was stimulated in BET-exposed fish of the F0 generation. F0 males exposed to DHAA and F0 females exposed to BET showed lower plasma Vtg concentration, but F1 males exposed to BET showed an increase in Vtg. In fish exposed to E2, the positive control for estrogenic effects, a pronounced increase in Vtg concentration was observed. Plasma sex steroids were not significantly affected by the wood extractives. However, although not statistically significant, the T concentration tended to be lower in fish of all BET treatments. The histological study revealed alterations in spermatogenic stages of F0 males exposed to DHAA and BET, which were different from those caused by E2. In F1 females, the percentage of vitellogenic oocytes was decreased in DHAA, BET and E2 exposures. This study shows that DHAA and BET may contribute to growth alterations and reproductive disturbances reported in fish exposed to pulp and paper mill effluents. Further, these wood extractives may have different effects in F0 and F1 generation fish, which highlights the value of two-generation studies in investigations regarding endocrine disrupting compounds.

In vivo and in vitro effects of flutamide and diethylstilbestrol on fetal testicular steroidogenesis in the rat.

Prenatal testosterone surge is considered crucial for physiological masculinization of male progeny. Disorders in sex steroid hormone balance during the fetal development may interfere with male reproductive health later in life. In this study, we have investigated in utero and in vitro effects of flutamide (FLU) and diethylstilbestrol (DES) on fetal rat testicular steroidogenesis. In utero exposure to FLU 25mg/kg or DES 0.02mg/kg had no obvious effects on ED 19.5 rat testicular testosterone and progesterone production, StAR protein or AR protein expression. However, when ED 19.5 rat testis were cultured for 180min in the presence of 0.1, 1, 10 and 100mg/l of FLU or DES, the highest doses of both compounds were capable of disturbing steroidogenesis. To study the rate of the changes seen in testicular steroidogenesis after 180min, time-series experiments, in which intact testes were cultured with FLU 100mg/l or DES 100mg/l for 30, 60 or 120min, were performed. In vitro time-series experiments revealed that changes in steroidogenesis occur very fast. Experiments with FLU brought further evidence to the hypothesis that ARs have negative autocrine role in developing Leydig cells.

Inhibition of tyrosine kinases PDGFR and C-Kit by imatinib mesylate interferes with postnatal testicular development in the rat.

The tyrosine kinase receptor c-kit and its interaction with the ligand, stem cell factor (SCF), play an essential role in the developing testis. C-kit is important for the development of the Leydig cells and for the migration, proliferation and survival of spermatogonia. Platelet-derived growth factor (PDGF) and its tyrosine kinase receptor (PDGFR) are important for the development of Leydig cells and myoid cells. The chemotherapeutic agent, imatinib mesylate (STI571, Glivec; Novartis) inhibits both of these tyrosine kinase receptors. Three-day treatment of immature male rats (SD) with imatinib (150 mg/kg) on postnatal days 5-7 delayed the formation of germ-line stem cell pool, reduced proliferation of type A spermatogonia and induced germ cell apoptosis. PDGFR-mediated proliferation of mesenchymal myoid precursors was also decreased and the length of the seminiferous cord was reduced. However, at the age of 11 weeks the exposed animals had normal epididymal sperm counts, whereas plasma levels of luteinizing hormone and follicle stimulating hormone were significantly increased. Imatinib serves as a good tool to study postnatal formation of the male germ-line stem cell pool and factors determining the final testicular size. As development of the human testis is controlled by the same mechanisms, further studies with primate and human models are needed to explore whether imatinib affects the testis in children as well.

Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on rat follicular steroidogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant and causes adverse effects on female reproduction when administered to rats. Our aims were to study effects of gestational and lactational exposure to TCDD on ovarian steroidogenesis and steroidogenic enzyme expression of offspring on postnatal day (PND) 14 in the rat and sensitivity of enzymatically isolated ovarian follicles to TCDD in vitro. Synthetic estrogen diethylstilbestrol (DES) was used as a treatment control. Serum progesterone (P4) level in offspring increased significantly on PND 14 in the TCDD (1 microg/kg)-exposed group while body weight, FSH and E2 levels were not changed. In ovarian follicles of offspring on PND 14 in the TCDD-exposed groups, protein expression of cytochrome P-450 aromatase, cytochrome P-450 cholesterol side-chain cleavage, steroidogenic acute regulatory protein, 3beta-hydroxy-steroid-dehydrogenase/Delta(5)-Delta(4) isomerase type 1, or P4 receptor was not affected. TCDD decreased E2 and P4 production in ex vivo follicle culture. DES at a dose level of 0.1mg/kg was dystocic while a dose 0.02 mg/kg increased ovarian ex vivo E2 and testosterone production without affecting P450arom activity indicating stimulation of early steps of steroidogenic pathway. Data suggests that TCDD has multiple targets in ovarian steroidogenesis, but the inhibitory action represented as decreased follicular steroid hormone production ex vivo is not apparent at the ovarian protein expression. Furthermore, TCDD had no direct effect on immature rat ovarian steroidogenesis in vitro suggesting that the follicle culture method is not a sensitive method to study the mechanisms of TCDD action.

Division of the male rat rhabdosphincter into structurally and functionally differentiated parts.

In order to understand the structure-function relationship in the male rat rhabdosphincter, the 3D structure of the striated muscle and associated dense connective tissue was reconstructed from representative serial sections cut from the proximal urethra harboring the muscle. The 3D structure was correlated with electromyography (EMG) of the rhabdosphincter, urodynamic parameters (bladder pressure and flow rate), and longitudinal contraction force of the proximal urethra. The muscular component of the rhabdosphincter consisted of a homogeneous population of the fast-twitch-type fibers. In the cranial part, striated muscle formed a complete ring encircling the urethra, deferent ducts, and ducts from seminal vesicles and prostatic lobes. Toward the middle part, the amount of densely packed connective tissue lacking type III collagen increased anteriorly and posteriorly and penetrated the muscular ring that became divided first posteriorly and then anteriorly into two symmetrical halves. In the caudal part, a thin midsagittal dense connective tissue septum remained posteriorly. EMG recordings suggested that the rhabdosphincter muscle was functionally divided into two parts. Unlike the cranial and middle parts, the caudal part did not show the first depolarization peak. It appears that rapid oscillatory oblique-to-circular muscular contractions proceeding in craniocaudal direction in the cranial and middle part draw the anterior wall supported by arch-like dense connective tissue closer to the posterior wall supported by a more rigid rhomboidal raphe. Longitudinal contractions of the urethra are possibly evoked from the proximal and caudal parts of rhabdosphincter. These could lead to simultaneous increase in urethral pressure ensuring rapid urine flow rate. The caudal part could augment the opening of urethral lumen during oscillatory voiding.

Effects of neonatal exposure to 4-tert-octylphenol, diethylstilbestrol, and flutamide on steroidogenesis in infantile rat testis.

Exposure of neonatal testis, populated by fetal-type Leydig cells, to endocrine-active compounds may have far-reaching consequences. Our aim was to resolve the sensitivity of testosterone synthesis of infant rat (Sprague-Dawley) testis to diethylstilbestrol (DES; 0.1-1.0 mg/kg), 4-tert-octylphenol (OP; 10-100 mg/kg), and Flutamide (FLU; 2.0-25 mg/kg) given by daily sc injections from birth to postnatal day 4. Testes and serum were collected on day 14 when body and testis weight, testicular histology, circulating testosterone, LH and FSH levels, and steroidogenic acute regulatory protein (StAR) and 3beta-hydroxy-steroid-dehydrogenase (3beta-HSD) protein levels were determined. DES at each dose and FLU at 25 mg/kg dose reduced testis weight and the diameter of seminiferous cords. FLU caused some Leydig cell hyperplasia. Plasma testosterone was reduced in all DES animals, LH elevated in DES 0.5 mg/kg and FLU 25 mg/kg animals, and FSH reduced in the DES 1.0 mg/kg group. Basal testicular ex vivo progesterone and human chorionic gonadotropin (hCG)-stimulated testosterone production were decreased in DES animals. Despite a decrease in hCG-induced cyclic adenosine-3',5'-monophosphate (cAMP) production, intratesticular testosterone was increased in the FLU 10 and 25 mg/kg groups. OP 100 mg/kg elevated hCG-induced progesterone production only. No changes were seen in 3beta-HSD protein levels in any treatment group. StAR levels were reduced in DES animals. The results indicate the sensitivity of postnatal fetal-type Leydig cells to endocrine-active compounds. Suppression of StAR expression level was an early sign of the DES-induced steroidogenic lesion. FLU-induced changes suggest the importance of androgen receptor-mediated regulation of testosterone synthesis in the postnatal rat testis. Octylphenol appeared less effective in bringing about acute steroidogenic changes.

In vitro effects of diethylstilbestrol, genistein, 4-tert-butylphenol, and 4-tert-octylphenol on steroidogenic activity of isolated immature rat ovarian follicles.

Isolated rat ovarian follicles grow and produce steroid hormones in vitro and so provide a good model for studying the effects of hormonally active compounds on follicular steroidogenesis. We have evaluated the effects of diethylstilbestrol (DES), genistein (GEN) and two alkylphenols, 4-tert-butylphenol (BP) and 4-tert-octylphenol (OP) on the growth, survival, and steroid hormone and cAMP production by isolated 14-day-old rat (Sprague-Dawley) ovarian follicles. During a 5-day culture, FSH was obligatory for follicle growth and increased estradiol and testosterone secretion in a dose-dependent manner. DES (10(-6) M) caused the strongest decline in estradiol and testosterone levels but did not have detectable effects on either cAMP production or aromatase enzyme activity. GEN caused a prominent decrease in cAMP and testosterone levels without significant changes in secreted estradiol. The latter, apparently, was due to a dose-dependent stimulation of aromatase enzyme activity in the presence of genistein. Both BP and OP decreased estradiol and testosterone secretion in a dose-dependent manner while no effect on aromatase activity was observed. OP, unlike BP, decreased forskolin-induced cAMP levels. Xenoestrogens at the used concentrations did not interfere with the growth and survival of the follicles. The results indicate that isolated ovarian follicles representing intact morphological and functional units offer a sensitive model system for elucidating the female-specific reproductive effects of environmental chemicals.

Pattern of male reproductive system effects after in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in three differentially TCDD-sensitive rat lines.

Male reproductive effects induced by in utero and lactational exposure to TCDD were analyzed in three rat lines that are differently sensitive to TCDD. Rats from lines A, B, and C were selectively bred from TCDD-resistant Han/Wistar (Kuopio, H/W) and TCDD-sensitive Long-Evans (Turku/AB, L-E) rats and exhibited very different LD50 values for TCDD: >10,000, 830, and 40 microg/kg in males, respectively. The resistance in line A rats was linked to a mutated H/W-type aryl hydrocarbon receptor (Ahr(hw)) and in line B rats to a H/W-type unknown allele B (B(hw)). Line C rats had no resistance alleles. Influence of the resistance alleles on developmentally induced male reproductive effects of TCDD was studied by exposing pregnant females to TCDD (0.03, 0.1, 0.3, or 1 microg/kg) on gestation day (GD) 15. Male progeny were sacrificed on postnatal day (PND) 70. Next, the dams were given 1 microg/kg TCDD on GD 15 and male progeny were sacrificed on PND 14, 21, 28, 35, or 49. Serum testosterone concentration, male sex organ weights, and testicular and cauda epididymal sperm numbers were analyzed; the most sensitive end point was decreased sperm numbers. The dose of 1 microg/kg TCDD reduced daily sperm production by 9.3, 25, and 36%, and cauda epididymal sperm reserves by 18, 42, and 49% in rat lines A, B, and C when measured on PND 70, respectively. The most consistent and significant effect was decreased weight of prostate lobes. The growth of the male reproductive organs was not markedly affected by the resistance alleles Ahr(hw) and B(hw). In contrast, the effects on sperm parameters appeared to be slightly modified by the resistance alleles. Thus, the intraspecies genetic differences in C-terminal transactivation domain of AHR appear to modify the sensitivity to only certain dioxin-induced male reproductive effects.

Effects of 4-tert-octylphenol, 4-tert-butylphenol, and diethylstilbestrol on prenatal testosterone surge in the rat.

In the present study, we evaluated the effects that 4-tert-octylphenol (OP) and 4-tert-butylphenol (BP) had on the prenatal testicular testosterone surge at embryonic day (ED) 19.5 in the rat. In utero exposure to alkylphenols (0.1-100 mg/kg maternal weight) on EDs 13.5, 15.5, and 17.5 did not decrease testicular testosterone content, whereas exposure to diethylstilbestrol (DES) caused a significant depression in testosterone synthesis and secretion. The depression was maintained during ex vivo tissue culture. In order to elucidate the observed differences in the in vivo effects between alkylphenols and DES, the exposures were also carried out in tissue culture of intact ED 19.5 testes. Basal testosterone, progesterone, cAMP production and hCG-induced testosterone levels were determined during and after a 3-h culture period. DES (100 mg/l) did not alter testosterone production but caused a two-fold increase in progesterone. OP (10, 100, 500 mg/l) and BP (100 mg/l) significantly increased testosterone and progesterone levels by up to seven-fold. In the presence of BP 100 mg/l, however, the intratesticular testosterone content did not correlate with the significantly increased fraction of secreted, or leaked, testosterone. The latter was correlated with tissue damage observed at electron microscopic level. Consistent with this, BP 500 mg/l elevated testicular testosterone level slightly during the first hour in the culture but the level subsequently returned to the control value. At the electron microscopic level, alkylphenols caused most severe changes in Leydig cell membrane structures and lipid droplets. In the DES-treated testes, membrane vesicle formation around the lipid droplets and increased mitochondrial pleiomorphy were observed. Altogether, the present in vivo and in vitro analyses confirm different effects of alkylphenols and DES on fetal rat steroidogenesis and tissue structure.

Microtubule-associated epithelial protein E-MAP-115 is localized in the spermatid manchette.

A microtubule-associated protein E-MAP-115 has been originally isolated and characterized from HeLa cells. Because of its predominant expression in cultured cells of epithelial origin, it has been suggested to be involved in the regulation of cell polarization. The present immunocytochemical, Northern blot and in situ hybridization analysis of E-MAP-115 in the mouse and rat seminiferous epithelium indicates its distinct association with the spermatid manchette, a unique microtubular structure which appears in the cytoplasm of spermatids at step 8 when nuclear polarization and elongation starts. At steps 15-16 when manchette has been disassembled, immunoreactivity for E-MAP-115 disappeared. At immunoelectron microscopical level, E-MAP-15 was associated with the microtubules of the manchette. In the Western and Northern blot analysis, a distinct stage-dependent expression of a single E-MAP-115 polypeptide and two mRNA species (3.4 and 2.4 kb) could be identified. MTEST 60, a spermatid-specific transcript, showed a 100% homology over region of 68-193 bp of E-MAP-115 sequence. The reported specific localization of E-MAP-115 to the spermatid manchette strongly supports its role as a regulator of cell polarization. This, in turn, supports the hypotheses concerning the dynamic function of the manchette during spermiogenesis.