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Herbs and spices have been used throughout human history for their medicinal qualities. The advent of cheap and readily available medicines have lessened the need for herbs and spices as traditional medicines, however, they are rapidly regaining popularity with rising interest of the general population in health, natural products and nutrition. The need for alternative medicines with antimicrobial properties, such as herbs and spices, has also come to the forefront in light of the recent bans of antibiotic use in the livestock industry, including the poultry industry. This large scale use presents an opportunity to observe nutrigenomic effects of prolonged use of herbs on a substantial number of birds fed high concentrations of these products throughout the production cycle. In this manuscript, we investigated the transcriptional effect of continual prolonged oregano supplementation on chicken ileum gene expression. Based on ileum transcriptomics, we report that continual supplementation with 2% oregano altered microbiota-gut-brain axis signalling, rearranged cancer susceptibility towards reduced steroid hormone-related cancers and altered expression of genes targeted by many registered drugs, thus likely affecting their efficiency and side effects. Transcriptional toxicology analysis indicated significant activation of Ventricular Septal Defect and Congenital Heart Disease categories. Our results, counter the notion that natural products such as oregano have the potential for little to no side-effects as they are “natural”. The nutrigenomic approach of understanding benefits and side effects of the food we eat, can revolutionize disease management and therapy and have special significance in designing the diets for individuals or livestock with known disease predispositions.

Overt and subclinical maternal hypothyroidism affects cartilage and bone extracellular matrix formation during fetal endochondral bone development. Less is known about the effect maternal hypothyroidism has on cell proliferation and death of growth plate chondrocytes. This study aimed to evaluate the expression of proliferation (Ki67), autophagy (LC3) and apoptosis (caspase 3) markers in the epiphyseal cartilage of the tibia in pups from subclinical and overt hypothyroid does, in their early postnatal development. Hypothyroidism was induced with propylthiouracil using low (1.5 mg/L) for subclinical (H1) and high dose (150 mg/L) for overt (H2) form. Propylthiouracil was administered through drinking water in pregnant Albino Oxford does from the first day of gravidity and during lactation. Control (C) group was not treated. Six, seven-day-old male pups from each group were euthanized. Histological examinations were performed on paraffin sections of the proximal tibial growth plate. Immunohistochemistry was used to assess the expression of Ki67, LC3 and caspase 3. The number of Ki67 positive chondrocytes was evaluated in the resting and proliferating zone using Image J. The expression of LC3 was performed by counting the number of positive dots in chondrocytes. The number of Ki67 positive chondrocytes was higher in both hypothyroid groups, while the number of LC3 positive dots per cell was lower indicating that the chondrocyte cell cycle in hypothyroid animals is faster and that the basal level of autophagy is reduced due to frequent mitosis. Compared to controls, caspase 3 expression in the terminal hypertrophic chondrocytes was reduced in H1 and completely absent in H2 pups which might indicate impaired differentiation. We conclude that both forms of maternal hypothyroidism in rats lead to accelerated proliferation, slowed autophagy and compromised apoptosis of terminal hypertrophic chondrocytes in the early infantile period and a delay in cartilage to bone transitio

The stimulative effects of thyroid hormones on mitochondria are realized through both non-genomic and genomic mechanisms, affecting respiration, mitochondrial plasticity and biogenesis. The subclinical form of maternal hypothyroidism in rats induces significant reduction of mitochondria number but also an augmentation of their area in neonatal and early infantile offspring dyctiotene oocytes. This study aimed to investigate if this form of subclinical hypothyroidism affects mitochondrial morphology and distribution in the early prophase of meiosis I oocytes. It was performed on newborn control (C) (n=10) and hypothyroid (SCH) (n=10) female rat pups derived from control (n=6) and propylthiouracil treated pregnant dams (n=6), respectively. Ovaries of all pups were removed and processed for transmission electron microscopy. The morphological features of mitochondria in the early prophase I oocytes until dyctiotene were assessed. No substantial differences were found in leptotene and zygotene oocytes in SCH group comparing to control, except just a few mitochondria characterized with shortened cristae, presence of wide pale area centrally positioned and membrane disruption. Pachytene mitochondria in treated pup oocytes were in great extent with disrupted membrane, shortened cristae and wide pale area centrally positioned while these features were rarely observed in control ones. Our results confirm altered mitochondria morphology found in primordial and primary follicles in case of maternal hypothyroidism, indicating their impaired function and possibly, propensity to programmed cell death. Further investigations may indicate to what extent pachytene, as a meiotic checkpoint, appears to be a milestone possibly predetermining the future of the cell.

Maternal hypothyroidism in its overt form affects skeletal development of the offspring, but these data are not available for the subclinical form which is becoming very frequent among pregnant women. We hypothesized that the subclinical form of hypothyroidism in rat dams, influences the process of offspring endochondral ossification affecting proliferation and differentiation of chondrocytes, osteoclasts and osteoblasts in pups. Seven-day-old male pups (n=18) derived from control dams and dams treated with a low dose (1.5 mg/L) or high dose (150 mg/L) of propylthiouracil in drinking water during pregnancy and lactation were used. Histomorphometric analysis of pups’ tibia proximal growth plate, expression of mRNA, immunohistochemical and histochemical visualization of extracellular matrix components was performed. The length of the tibia was reduced in hypothyroid pups. Secretion of type 2 and 10 collagens in the subclinical and overt form were lower while the amount of glycosaminoglycans was higher when compared with controls. Down-regulated tartrate resistant acid phosphatase mRNA indicated altered osteoclasts function while lower expression of dentin matrix acid protein–1 mRNA and reduced synthesis of type 1 collagen accentuated a compromised bone formation in the overt form of hypothyroidism. The subclinical form of maternal hypothyroidism had a negative effect on the differentiation of hypertrophic chondrocytes and calcified cartilage removal in 7-day-old pups. In addition, overt hypothyroidism had a negative effect on the proliferation of chondrocytes and deposition of osteoid. Both forms of hypothyroidism resulted in a decrease of tibia length due to changes in growth plate formation.

Introduction. The ovarian surface epithelium (OSE) undergoes intensive regeneration and remodeling after each ovulation during the whole reproductive period. This process increases the risk of one of the most common ovarian tumors in women and the female dog. Considering the fact that maternal hypothyroidism highly impacts cell proliferation and cell death during folliculogenesis in the early neonatal period, we aimed to analyze its effect on OSE morphology and dynamics. Materials and Methods. The study was performed on newborn (24-h-old) and neonatal (4-day-old) female rats, a randomized trial between the control and hypothyroid groups, born under controlled circumstances and hypothyroid mothers, respectively. Their ovaries were analyzed histologically and processed to determine the OSE cell height as an average value of four measurement points. Also, the immunopositivity of the proliferating cell nuclear antigen (PCNA) and caspase-3 were assessed semiquantitatively. Results and Conclusions. No major structural differences of OSE were found between groups within the given ages except for a slight increment of OSE cell height and incompleteness of apical cell membrane with cytoplasmic projections in hypothyroid animals. PCNA immunopositivity of the OSE cells was higher in ovaries of hypothyroid animals of both ages in comparison to the controls. Moreover, only scarce OSE cells were caspase-3 positive in both groups and ages, with no difference in immunopositivity. Our study confirms the impact of hypothyroidism in the early postnatal period onmorphology and proliferation rate of OSE cells, with no effect on caspase-3 dependent cell removal, which may serve as a premise for future investigation of potential carcinogenesis, in terms of prevention and treatment of ovarian cancer.

The effects of maternal hypothyroidism on the development and function of different organs of their offspring are well known. It is unclear how its subclinical form affects some of these processes, including endochondral ossification during prenatal and early postnatal development. Our previous studies have demonstrated that hypothyroid rat pups had reduced growth plate thickness. The aim of the study was to assess bone turnover by defining the expression of tartrateresistant acid phosphatase (TRAP), alkaline phosphatase tissue nonspecific isozyme (ALPL) and mRNA for dentin matrix acidic phosphoprotein (DMP1) in the tibia of pups from subclinical hypothyroid dams in early postnatal development. Subclinical hypothyroidism (SHT) was induced with propylthiouracil through drinking water (1.5 mg/l) in pregnant Albino Oxford dams from the first day of gravidity and during lactation. The control group was untreated. Five, sevenday- old pups from each experimental group were euthanized. All histological examinations were performed on paraffin sections of the proximal tibial growth plate. Histochemistry was used to assess the expression of TRAP, immunohistochemistry for ALPL and in situ hybridization for DMP1. The area of the TRAP positive signal, optical density of ALPL immunostaining, and the number of DMP1 positive cells were evaluated using the Image J. Expression of TRAP and ALPL in the proximal tibial growth plate were lower in the hypothyroid pups, and the number of cells expressing mRNA for DMP1 was decreased. TRAP is a marker of osteoclast activity. Osteoclasts are multinucleated cells which are necessary for bone and mineralized cartilage resorption. In osteoblasts ALPL and DMP1 are expressed and they have an important role in the process of endochondral ossification. As we have shown that TRAP, ALPL and DMP1 are decreased, we may conclude that maternal SHT in rats leads to compromised cartilage to bone transition, and delayed osteogenic differentiation in the earl

The ovarian surface epithelium (OSE) is a very dynamic part of the ovary, with an important role in ovulation during the reproductive period. It represents a source of germ cells in the fetal period but data regarding the role of the OSE in the postnatal period are contradictory. Although the OSE has been the subject of research in the past few years, its ultrastructure in newborn rats has not yet been studied. The aim of this study was to examine rat OSE ultrastructure features in the first 24h hours after birth. Six newborn rats were euthanized at 12h±2 hours after birth. Their ovaries were fixed in 4% glutaraldehyde, post-fixed in 1% osmium tetroxide, routinely dehydrated and embedded in araldite. Semi-thin sections were stained with toluidine blue, and used to select areas of interest. Ultra-thin sections of selected areas were mounted on copper grids and examined under a Philips CM12 transmission electron microscope. Ultrastructural analysis revealed the presence of a typically single-layered OSE with sporadic pseudostratified appearance, both consisting of cuboidal and/or columnar cells. The OSE cells’ nuclei had irregularly dispersed chromatin, and a central or apical location. From the surface of the OSE cells numerous microvilli and cytoplasmic projections extended towards the peritoneal cavity. Continuous tunica albuginea was not found, but occasionally groups of fibroblast-like cells were present, indicating the onset of its formation. Numerous oogonia and oocytes were identified between OSE cells or up to their basal part, suggesting that extrusion (the release of germ cells into the peritoneal cavity) may be an important mode of germ cell elimination. In the early postnatal period in rat ovaries massive death takes place of oogonia and oocytes, mostly via apoptosis and autophagy. Further investigation might reveal the contribution of extrusion as another form of germ cell elimination for both, maintaining and balancing their number in the ovary, which i

Thyroid hormones (TH) play a pivotal role in the development of mammalian skin, and are necessary for both the initiation and maintenance of hair growth. Adult hypothyroid rats showed impaired epidermal proliferation, hair growth and wound healing. Stem cells (SCs) located at the bulges of the hair follicles are responsible for hair cycling, and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of bulge stem cells could be a cause of disturbed skin reepithelization and hair follicle maintenance. We hypothesized that a decrease in TH during prenatal development would affect the bulge stem cell population. Subclinical hypothyroidism was induced with propylthiouracil through drinking water in a dose of 1.5 mg/l in pregnant Albino Oxford rats, from the first day of gravidity and during lactation. The control group was untreated. Five, seven-day-old pups were euthanized and skin samples were taken from the dorsal part of their bodies. The number of hair follicles was estimated on an area of 1mm2 of dermis. For the immunohistochemical study, proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology) and NANOG (Thermo Scientific) antibodies were used. The number of PCNA and NANOG positive cells was estimated on the area of 1mm2 of hair follicle. The number of hair follicles was reduced and the expression of PCNA was decreased in the cells of the inner and outer sheath of hair follicles in the hypothyroid pups compared to the controls. They also had an increased number of NANOG positive bulge cells which we presume are multipotent SCs. Decreased expression of PCNA demonstrates low proliferation and increased expression of NANOG, which may be explained in two ways: 1) the inhibited differentiation of putative SCs and/or 2) the reduction of mobilization of these cells to the epidermis or sebaceous gland. Our results confirm that the lack of TH influences the formation of hair follicles in early infant

Uvod: Poznato je da su tireoidni hormoni (TH) od esencijalnog značaja za razvoj, funkciju i diferencijaciju svih ćelija u organizmu u toku prenatalnog i postnatalnog života. Prethodna istraživanja dovode u vezu smanjen nivo TH i razvoj reumatoidnog artritisa i drugih oboljenja zglobova, međutim mehanizam nastanka navedenih promena još uvek nije razjašnjen u potpunosti. U sinovijalnim membranama (SM) zglobova zahvaćenih reumatoidnim artritisom, utvrđeno je povećanje broja ćelija koje eksprimiraju PCNA (proliferating cell nuclear antigen). Cilj ovog istraživanja je bio da se definiše uticaj subkliničkog hipotireoidizma majki tokom graviditeta na SM novorođenih jedinki, s obzirom da u dosadašnjim istraživanjima ovaj efekat nije dovoljno proučen. Materijal i metode: Subklinički hipotireoidizam izazvan je kod gravidnih jedinki Albino Oxford pacova, dodavanjem propiltiouracila kroz vodu za piće (1,5 mg/l) počevši od prvog dana graviditeta i tokom laktacije. Kontrolna grupa pacova nije tretirana. Mladunci su žrtvovani sedmog dana nakon rođenja. Sva histološka ispitivanja sprovedena su na parafinskim presecima debljine 5µm obojenim standardnim hematoksilin/eozin histohemijskim bojenjem, čija je analiza sprovedena na svetlosnom mikroskopu (Olympus BX53). Ekspresija PCNA je pokazana inunohistohemijski (Santa Cruz Biotechnology). Kvantifikacija ekspresije PCNA izvršena je pomoću softvera Image J. Rezultati: Pregledom preseka bojenih hematoksilin/eozinom nisu uočene značajne razlike u građi SM između kontrolne i tretirane grupe. Broj ćelija koje eksprimiraju PCNA u SM tretiranih životinja je značajno veća od broja ćelija u SM kontrolnih životinja. Zaključak: Povećana ekspresija PCNA ukazuje na intenziviranu proliferaciju ćelija SM novorođenih pacova starosti 7 dana. Ovaj rezultat može biti osnova za nastavak istraživanja uticaja hipotireoidizma na razviće SM.

Uvod: Tireoidni hormoni (TH) imaju važnu ulogu u postnatalnom razvoju i očuvanju funkcije skeletno zglobnog sistema. U procesu endohondralnog okostavanja, TH svojim delovanjem na hondrocite podstiču rast kostiju. Brojna istraživanja su pokazala efekte hipotireoidizma na ovaj proces na različitim eksperimentalnim modelima, a cilj ovog istraživanja je bio da definiše uticaj subkliničkog hipotireoidizma majki u toku graviditeta i laktacije na proces endohondralnog okoštavanja tibije mladunaca starih sedam dana. Materijal i metode: Subklinički hipotireoidizam izazvan je kod gravidnih jedinki Albino Oxford pacova, dodavanjem propiltiouracila u vodu za piće (1,5 mg/l), od prvog dana graviditeta i tokom laktacije, dok kontrolna grupa nije tretirana. Mladunci su žrtvovani sedmog dana nakon rođenja. Histološka ispitivanja sprovedena su na 5µm debelim parafinskim presecima tibije bojenim hematoksilin/eozinom. Visina proksimalne epifizne ploče merena je na presecima na svetlosnom mikroskopu (Olympus BX53), uz odgovarajući softver SellSensEntry (Olympus). Ekspresija kolagena X (ColX) je pokazana in situ hibridizacijom. Kvantifikacija ekspresije ColX izvršena je pomoću softvera Image J. Rezultati: Visina epifizne ploče je značajno niža kod mladunaca iz tretirane grupe u poređenju sa kontrolnom grupom. Merenjem površine koju zahvata pozitivan signal u odnosu na ukupnu površinu zone hipertrofičnih hondrocita utvrđeno je značajno smanjenje ekspresije ColX kod jedinki iz tretirane grupe. Zaključak: Ovi rezultati ukazuju da subklinički hipotireoidizam majki za vreme graviditeta remeti hipertrofičnu diferencijaciju hondrocita i fiziološki razvoj epifizne ploče.

The purpose of this work was to investigate the infl uence of hypothyroidism on spleen tissue morphology and immune cell density in fourteen-day-old juvenile rats. Hypothyroidism in pups (n=10) was induced by administration of propylthiouracil (PTU) in drinking water (1.5 mg/L) to their mothers during pregnancy and period of lactation. Fourteen-day-old pups were sacrifi ced and the thyroid-stimulating hormone (TSH) serum concentration and thyroid activation index (Ia) were determined. Increased serum level of TSH and increased Ia showed that pups from PTU treated mothers were hypothyroid. White and red spleen pulp, marginal zone and connective tissue volume density has been assessed by using the stereological method. Using immunohistochemistry, the present CD3+ T lymphocytes, CD45RA+ B lymphocytes and CD68+ macrophages were quantifi ed. A signifi cant reduction of volume density of the periarteriolar lymphocyte sheath (VvPALS) and lymphatic follicles (Vvf) due to depletion of T and B lymphocytes respectively, was observed in the spleens of hypothyroid pups compared to controls. The volume density of the red pulp (Vvrp), marginal zone (Vvmz) and connective tissue (Vvct) was increased, as well as the number of CD68+ macrophages in the spleens of hypothyroid pups compared to controls. These results indicate that thyroid hormones might be important for normal development of both, specifi c and innate immune cells in the spleen during prenatal and early postnatal period.

Background: The aim of our study was to examine the effect of mild maternal hypothyroidism on the apoptosis of the oocytes in the ovaries of rats in the early postnatal period during formation of oocytes and follicles. Materials and Methods: Hypothyroidism was induced in pregnant and lactating rats by feeding 1.5mg/L propylthiouracil (PTU) through drinking water. Ovaries were obtained from newborns on postnatal day (PND) 0, 4 and 7. The degree of oocyte apoptosis was measured from the largest cross sections (n=2) from each ovary using by Colorimetric - TdT (terminal deoxynucleoid transferase) Enzyme in situ cell death detection kit (Merck). Results: This preliminary result suggests that hypothyroidism induce increase of the total oocyte number from 4. day in the experimental group while the number of apoptotic oocytes is the lowest that day. However, the highest percentage of apoptosis is noticed on day 0. in control group. Conclusion: In our further investigation we will notice whether the morphological changes induced on the oocytes appear before or after development of primordial follicles as a result of the impacts of maternal hypothyroidism on folliculogenesis of their offspring.