Archiwa

1. Research Institute of Animal Production, Nitra, Slovak Republic, prof. Alexander V. Sirotkin
2. Department of Animal Science, University of Kentucky, Lexington, USA, prof. William J. Silvia
3. Banat’s University of Agricultural Sciences and Veterinary Medicine, Timisoara, Rumunia, prof. Horia Cernescu

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In our studies, it has been demonstrated:

• The importance of adrenergic stimulation in the corpus luteum function;
• The mechanism of autoregulation of corpus luteum function ;
• Non-genomic effect of steroids on the uterus and ovary function;
• The disturbing effect of xenobiotics on the function of reproductive tract, through the genomic and non-genomic way.

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1. J.Kotwica – Effect of organochlorine xenobiotics on state of maternal-foetal connections in pregnant cows.
2. M.Kowalik – The role of steroid hormones and lutetropic and luteolytic factors in regulation of the expression and function of membrane progesterone receptors in bovine reproductive tract.

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Applied methods

1. In vivo methods

• measurement of blood flow in the uterine artery of the pig by an electric transducer using an electromagnetic flowmeter (RT500 Narcomatic) under  general anaesthesia
• measurement of porcine uterine intraluminal pressure/indirect uterine contractile activity (Bridge Amp AD Instruments) under  general anaesthesia
• biotechnology methods of reproduction in the sheep: synchronization of ovulation (intravaginal suppositories) and acceleration of the breeding season (melatonin implants) for experimental purposes
• mid-ventral laparotomy of the pig and sheep: ovariectomy, hysterectomy, induction of ischemia in the uterus of the pig by ligation of the uterine artery, intravital collection of uterine samples, implantation of catheters to blood vessels of mesometrium for blood collection and/or infusion of biologically active substances e.g. hormones under  general anaesthesia
• implantation of catheters into blood vessels of the head and neck in the pig, sheep and rabbit under  general anaesthesia
• long-term collection of cerebrospinal fluid from the third brain ventricle in sheep
• long-term blood collection from jugular veins of the pig and sheep
• continuous monitoring of blood oxygen saturation, pulse rate data, carbon dioxide detection and visual respiration indicator using pulse oximeter with carbon dioxide detector (Nonin Medical Inc. 9847V) under general anaesthesia

2. Ex vivo methods

• the study of the contractile activity of the superficial veins of the head in the pig (frontal, nasal and facial veins)
• the study of the contractile activity of isolated arteries of the female reproductive tract in the pig (the uterine artery)
• the study on the isolated head of the pig, sheep, rabbit perfused with autologous blood
• the study on the isolated pig’s ovary and uterus, testicle of the pig and rat perfused with autologous blood

3. Tissue incubations

• pig’s hypothalamus explants

4. Analytic methods

4.1  Molecular biology methods

Expression on the mRNA level: Real Time PCR (qPCR) or Real Time PCR with TaqMan probes (qPCR TaqMan) and on the protein level: Western blot analysis (WB):

• factors associated with production of cerebrospinal fluid: VEGF and its receptors: Flt-1, Flk-1 and co-receptor NRP-1, aquaporin-1,  aquaporin-4, claudin-2 (qPCR, WB) in the ovine choroid plexus
• tight junction proteins restricting the passage of molecules via the intercellular path: transmembrane proteins: occludin, claudin-1, JAM-1 and cytosolic (scaffolding) proteins: ZO-1, ZO-2, ZO-3 and afadin-6 (qPCR, WB) in the ovine choroid plexus
• transport proteins: transtyretin (TTR) in the ovine choroid plexus (qPCR)
• lipopolysaccharide receptor complex: toll-like receptor 4 (TLR4), CD14 and MD-2 in the ovine choroid plexus (qPCR)
• cytokines and their receptors and chemokines: TNFα, IL-1β, IL-6, TNFRI, TNFRII, IL-1R1, IL-1R2, IL-6R, gp130, MCP-1 in ovine choroid plexus (qPCR)
• metalloproteinases and their tissue inhibitors: MMP2, MMP3, MMP9, TIMP-1 and TIMP-3 (qPCR) in the ovine choroid plexus
• hypoxia marker HIF-1α in in the porcine uterus (qPCR, WB)
• biological clock elements: Bmal 1, Clock, Npas2, Per 1-3, Cry 1-2 (qPCR TaqMan, WB) in the hypothalamus of domestic pig
• nuclear receptors associated to the functioning of molecular biological clock: Rev-erb α, Rev-erb β, Ror α, Ror β, Ror γ (qPCR TaqMan) in the hypothalamus of domestic pig
• antioxidant enzymes in the testis and epididymis of male deer and bison: EcSOD, GpX 4, GpX 5 (qPCR), SOD-1, Cat (qPCR, WB) and SOD-1, SOD-2 in porcine uterus (qPCR)
• leptin receptor (Ob-Ra i Ob-Rb) in the choroid plexus and hypothalamus of domestic pig (qPCR, WB)

4.2 Immunoenzymatic (ELISA) method:

• LPS Binding Protein in sheep
• IL-6 in sheep

4.3 Radioimmunoassay (RIA) in plasma, tissues and body fluids:

• thyroid hormones: T3 and T4 (free and total)
• melatonin
• hormones of reproductive system: estradiol, estrone, progesterone, testosterone, oxytocin, prostaglandin F2α
• protein hormones: prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH), leptin, activin A in pig

4.4 Labeling hormones with radioisotope

• oxytocin, GnRH, FSH, LH, prolactin, β-endorphin iodination (J¹²⁵)

4.5 Immunohistochemical localization (with fluorochrome):

• c-Fos protein (early response gene product) in porcine brain
• NADPH-diaphorase (histochemical method) in the blood vessels of porcine brain
• nitric oxide synthases, iNOS, eNOS in the porcine uterus and periovarian vascular complex and in rat’s large intestine
• endogenous hypoxia marker (HIF-1α) in porcine oviduct and uterus
• antioxidant enzymes SOD-1 and SOD-2 in porcine female reproductive tract and mice’s large intestine
• Vascular Endothelial Growth Factor (VEGF) and its receptors Flt-1, Flk-1 in the ovine choroid plexus
• antiapoptotic protein cFLIP in porcine female reproductive tract
• apoptotic cells with TUNEL assay (Terminal Transferase dUTP Nick End Labeling) in porcine uterus and rat’s large intestine
• dopamine hydroxylase (DβH) in porcine head vessels (facial, nasal and frontal vein) and peri-ovarian vascular complex
• leptin receptor in porcine hypothalamus and choroid plexus
• lipopolysaccharide receptors complex: CD14 and TLR4 in choroid plexus in sheep

4.6 Optical density measurments:

• immunoreactivity measurement with Axio Vision Rel. 4.8.2 program

Equipment

1. Equipment for in vivo, ex vivo experiments:

• Inhaled anesthetic apparatus for large animals (KOMESAROFF MINI-KOM)
• Cardiomonitor (TEMED)
• Pulse oximeter with carbon dioxide detector (Nonin Medica Inc.)
• Set for experiments on isolated organs (UNIPER, Up-100, Hugo Sachs Elektronik-Harvard)
• Set for measurement of isometric tension isolated blood vessels or muscle tissues (HSE F30, type 372, Hugo Sachs Elektronik-Harvard)
• Infusion pumps
• Peristaltic pump (Elphan, ZALIMP)
• Electromagnetic transducers for blood flow measurement (Flowmeter Narcomatic RT500)
• Surgical drill (Esculap)

2. Western blot and nucleic acids analysis workplace:

1. Mini PROTEAN system with PowerPac basic power supply (electrophoresis system and wet transfer system, Bio Rad)
2. Hematological roller (Chemland)
3. Horizontal electrophoresis system Wide Mini Sub Cell (Bio Rad)
4. Laminar flow chamber (Polon)

3. Equipment for Radioimmunoassay:

• Gamma Counter (LKB)
• Liquid Scintillation Counter (Beckman)
• Radiometer with probe Rust-3

4. Basic lab equipment:

• Vacuum pump BioVac 225 (OmegaLab)
• Spectrophotometer (Beckman)
• Stereoscopic microscope
• Vortex (Biosan)
• Shaker (Biosan)
• Incubation chamber with shaker (Edmund Buehler)
• Magnetic stirrer (Biosan, IKA WERKE)
• Heated dry block  (OmegaLab)
• Water bath
• pH-meter (HANNA Instruments)
• Mini centrifuge (Biosan, StarLab)
• Centrifuge with cooling (MPW, Beckman)
• Electronic scales (RADWAG, MEDICAT)
• Ultra low freezer (SANYO)
• Fume cupboard (Polon)
• Ice maker (Scotsman)
• Manifold Machine (pipettor) Hamilton Microlab 1000

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1. Laboratory of Physiology of Reproduction and Behavior, French National Institute of Agricultural Research (INRA), French national Center for scientific Research (CNRS), Francois Rabelais University of Tours and National Studs, Nouzilly, France (Dr Jean-Claude THIERY and Dr Laurence DUFOURNY);
2. Research Unit for Animal Life Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan (Prof. Noboru MANABE);
3. Institute of Biophysics, Biological Research Centre Hungarian Academy of Sciences in Szeged, Hungary (Dr. Maria DELI);
4. Evestra Inc., San Antonio, USA.

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Main achievements of the Department concern the explanation of physiological mechanisms of local and central regulations of the reproductive processes in females.

In central nervous system:

In reproductive organs:

Original theory on mechanisms regulating the estrous cycle, with special regard of physiological ishemia of the endometrium effect on course of animal estrous cycle was published.

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1. The level of inducible subunit – HIF-1α of the Hipoxia Inducible Factor-1 in nuclear fraction of the porcine endometrium in the estrous cycle (Statutory Found 2013; principal investigator: S. Stefańczyk-Krzymowska);
2. Transport of dopamine along  the cranial nerves (III-VI) from the midbrain into venous blood of the cavernous sinus and its countercurrent transfer from the venous blood to the arterial blood supplying the brain in the rabbit (Statutory Found 2013; principal investigator: J. Muszak);
3. The energy status of the pig uterus in conditions of its variable blood supply (Project of National Science Centre 2011-2014; principal investigator: B. Wąsowska);
4. Effect of melatonin on choroid plexus response to immune stress in ewes (Project of National Science Centre OPUS 2012-2015; principal investigator: J. Skipor);
5. Effect of photoperiod and melatonin on aquaporines expression in the choroid plexus of brain ventricles in ewes (Project of National Science Centre PRELUDIUM 2013-2015; principal investigator: A. Szczepkowska);
6. Hypothalamic control of reproduction in sheep: seasonal modulation of proteins in CSF and choroid plexus (Polish –French project in the frame of POLONIUM 2013-2014, project coordinator  J. Skipor);
7. Localization and correlation of apoptotic, anti-apoptotic factors and antioxidative enzymes in the porcine uterus under normoxia and hypoxia condition in vivo (Bilateral Scientist Exchange Program between Polish Academy of Sciences and Japan Society for the Promotion of Sciences 2013;  project coordinator: B. Wąsowska);
8. Evaluation of safety and pro-health activity of home and commercial probiotic preparations: model research on animals with induced colitis” (Project of National Science Centre 2011-2014; principal investigator: E. Wasilewska; co-investigator: B. Wąsowska);
9. The effect of immune stress and interleukin-1b on melatonin secretion in ewes during short and long days – in vitro and in vivo studies (Project of National Science Centre SONATA BIS 2012-2015: principal investigator: A. P. Herman, The Kielanowski Institute of Animal Physiology and Nutrition of Polish Academy of Sciences, co-investigator: A. Szczepkowska, P. Gilun).

Projects completed

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1. RIA and EIA of hormones and prostaglandins;
2. Isolation and in vitro culture of:
   • luminal and stromal cells of the porcine endometrium,
   • myometrial cells of the uterus,
   • endothelial cells,
   • epithelial cells of the oviduct,
   • luteal cells,
   • granulosa cells,
   • trophoblast cells,
   • anterior pituitary cells,
3. Collection, in vitro culture, cryopreservation and non-surgical transfer of porcine embryos;
4. Histological analysis of the female reproductive tract;
5. Immunohistochemical and immunofluorescent staining of tissues and cultured cells;
6. Scanning and transmission electron microscopy;
7. Western blot analysis;
8. Isolation of DNA / RNA;
9. Quantitative and qualitative DNA/ RNA analysis;
10. Real-time PCR;
11. Southern blot analysis;
12. Cell transfection with plasmid vectors.

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