Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis:an in vitro study

BACKGROUND: The cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression. METHODS: Uterine tissues were collected on Days 10–12 and 14–16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry. RESULTS: The results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P(4), E(2), AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus. CONCLUSIONS: This study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs

Zastosowanie hodowli i multiplex PCR w diagnostyce drobnoustrojów wywołujących zapalenie pochwy u kobiet z rakiem szyjki macicy

Background: Bacterial vaginosis (BV) and vaginitis in cervical cancer patients might becaused by mixed aerobic, anaerobic, and atypical bacteria. Since genital tract infections can be complicated, early and accurate identification of causal pathogens is vital. Objectives:The purpose of this study was i) to determinate if currently used aerobic culture methods are sufficiently sensitive to identify pathogens that can appear in the cervix of women after cancer treatment; ii) to investigate if molecular methods can improve the diagnostic process of BV and vaginitis, as well as broaden the range of detectable pathogens that would otherwise be difficult to cultivate. Methods: A one-year hospital-based study was conducted in 2011/2012. Cervical swabs from 130 patients were examined by microbiological culture and multiplex PCR. Results: Swab samples were positive for 107 and 93 women by microbiological culture and multiplex PCR, respectively. The most common bacteria isolated from culture were: Escherichia coli, Enterococcus faecalis, Streptococcus agalactiae, and Staphylococcus aureus, and using the molecular technique were: Gardnerella vaginalis, Bacteroides fragilis, Ureoplasma ureoliticum/parvum, Mobiluncus curtisii and Atopobium vaginae. Conclusions: Multiplex PCR might contribute to the diagnosis of genital tract infections and it broadens the number of detectable microorganisms responsible for BV. Combination of these two methods may become the basis for standardized diagnosis of BV and vaginitis.Bakteryjne zapalenie pochwy u pacjentów chorych na raka szyjki macicy może być spowodowane przez bakterie beztlenowe, tlenowe i atypowe. Ponieważ infekcje narządów płciowych mogą doprowadzić do poważnych komplikacji, wczesna i odpowiednia identyfikacja źródła infekcji jest bardzo ważna. Cel pracy: 1) określenie, czy obecnie używane metody hodowli tlenowej w pracowni mikrobiologicznej są wystarczające do identyfikacji patogenów, które mogą pojawić się w szyjce macicy po leczeniu nowotworowym; 2) zbadanie, czy molekularne metody mogą polepszyć diagnostykę bakteryjnych zapaleń pochwy i zwiększyć zakres wykrywanych patogenów o te, które są trudne w hodowli. Metodyka: Materiałem do badań były wymazy z szyjki macicy, pobrane od 130 kobiet z nowotworem szyjki macicy hospitalizowanych w Wielkopolskim Centrum Onkologii w latach 2011-2012. Identyfikacji mikroorganizmów dokonano tradycyjną metodą hodowlaną i metodą molekularną- multiplex PCR. Wyniki: Hodowla mikrobiologiczna zdiagnozowała 107 pozytywnych przypadków, zaś multiplex PCR- 93. Najczęściej izolowanymi patogenami metodą mikrobiologiczną były: Escherichia coli, Enterococcus faecalis, Streptococcus agalactiae i Staphylococcus aureus, a techniką molekularną: Gardnerella vaginalis, Bacteroides fragilis, Ureoplasma ureoliticum/parvum, Mobiluncus curtisii i Atopobium vaginae. Wnioski: Multiplex PCR mógłby pomóc w diagnostyce infekcji narządów płciowych i powiększyć zakres wykrywanych mikroorganizmów. Połączenie tych dwóch technik może stanowić podstawę standaryzacji diagnostyki zapalenia dróg rodnych kobiet

Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis: an in vitro study

BACKGROUND: The cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression. METHODS: Uterine tissues were collected on Days 10–12 and 14–16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry. RESULTS: The results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P(4), E(2), AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus. CONCLUSIONS: This study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs

Steroids affect gene expression, ciliary activity, glucose uptake, progesterone receptor expression and immunoreactive steroidogenic protein expression in equine oviduct explants in vitro

The oviduct undergoes dramatic functional and morphological changes throughout the estrous cycle of the mare. To unravel the effects of steroids on the morphology, functionality and gene expression of the equine oviduct, an in vitro oviduct explant culture system was stimulated with physiological concentrations of progesterone and 17β-oestradiol. Four conditions were compared: unsupplemented preovulatory explants, preovulatory explants which were stimulated with postovulatory hormone concentrations, unsupplemented postovulatory explants and postovulatory explants which were stimulated with preovulatory hormone concentrations. The modulating effects of both steroids on oviduct explants at different levels were investigated: 1) ciliary activity, 2) glucose consumption and lactate production pattern, 3) ultrastructure, 4) mRNA expression of embryotrophic genes, 5) steroidogenic capacities of oviductal explants, and 6) progesterone receptor expression. The present paper shows that the equine oviduct is an organ with potential steroidogenic capacities which is highly responsive to local changes in progesterone and 17β-oestradiol concentrations at the level of morphology, functionality and gene expression of the oviduct. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse

Steroids in the equine oviduct: synthesis, local concentrations and receptor expression

Steroids play an important role in mammalian reproduction and early pregnancy. Whereas systemic changes in steroid concentrations have been well documented, it is not clear how these correlate with local steroid concentrations in the genital tract. We hypothesized that, in the horse, the pre-implantation embryo may be subjected to high local steroid concentrations for several days. Therefore, we measured progesterone, 17-hydroxyprogesterone, 17β-oestradiol, testosterone and 17α-testosterone concentrations in equine oviductal tissue by means of ultra-high performance liquid chromatography coupled to tandem mass spectrometry and progesterone, 17β-oestradiol, oestrone and testosterone in oviduct fluid using radioimmunoassay, with reference to cycle stage and side of ovulation. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during diestrus, whereas other steroid hormone concentrations were not influenced by the side of ovulation. These results suggest that the high ipsilateral progesterone concentration is induced by 1) the contribution from follicular fluid in the oviduct and the diffusion of follicular fluid steroids after ovulation; 2) a local transfer of steroids via blood or lymph, 3) local synthesis of progesterone in the oviduct, as evidenced by the expression of steroidogenic enzymes and, 4) the paracrine contribution from follicular cells. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse

Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies