Uticaj Smanjene Funkcije Štitaste Žlezde Majki na Razvoj Jajnika Mladunaca Pacova u Ranom Postnatalnom Periodu

Abstract

Tiroidni hormoni (TH) su od ključnog značaja za razviće sisara u prenatalnom periodu. Prethodne studije pokazuju da subklinički hipotiroidizam ima za posledicu povećanu stopu atrezije u jajnicima mladunaca u prepubertalnom i pubertalnom uzrastu. Uticaj smanjene koncentracije TH na ranu postnatalnu folikulogenezu i razviće jajnika nije do sada ispitano. Ova disertacija ima za predmet istraživanja uticaj hipotiroidizma majki indukovanog 6-n-propil-2- tiouracilom (PTU) na folikulogenezu mladunaca pacova (n=15) 0, 4. i 7. dana po rođenju. Kod mladunaca 0. dana određivani su broj karakteristike oogonija i oocita normalne i izmenjene morfologije svetlosnom i elektronskom mikroskopijom. Pomenutim metodima kod mladunaca 4. i 7. dana starosti u jajnicima je određivan broj nestova, oogonija i oocita po nestu, primordijalnih, primarnih, sekundarnih i preantralnih folikula. Prisustvo glikogena i mukopolisaharida u tkivu jajnika mladunaca 0. dana određivano je PAS (eng. Periodic Acid Shiff) histohemijskim bojenjem, a prisustvo kalcijuma u tkivu jajnika pacova 4. i 7. dana Alizarin Red bojenjem. Volumenska gustina nukleusa, mitohondrija i glatkog endoplazmatičnog retikuluma u oocitama primordijalnih folikula analizirana je u jajnicima pacova 4. i 7. dana starosti. Imunohistohemijskom metodom detektovana je imunoekspresija PCNA (eng. proliferating cell nuclear antigen) kao markera proliferacije, dva markera apoptoze (kaspaza 3 i TUNEL (eng. terminaldeoxynucleotidyl transferase dUTP nick end labelling))kao i dva markera autofagijе (ATG7 i LC3I/II). Hipotiroidni pacovi na rođenju imali su smanjen broj oogonija u mitozi i u mirovanju, kao i povećan broj atipičnih oogonija/oocita. Na ultrastukturnom nivou pokazano je prisustvo degenerisanih ćelija u pahitenu (Z-ćelije), oogonija/oocita u autofagiji, apoptozi i kombinaciji ova dva tipa ćelijske smrti, kod obe grupe životinja ovog uzrasta. Najbrojniji tipovi atipičnih ćelija kod hipotiroidnih pacova 0. dana su bile oogonije/oocite u autofagiji i Z-ćelije. U prilog ubrzanoj maturaciji neizmenjenih oogonija/oocita kod ove grupe životinja ide zapaženo prisustvo prominentnih organela i ekspresija PCNA. Broj TUNEL pozitivnih oogonija/oocita je bio manji kod hipotiroidnih pacova 0. dana, dok nije bilo razlike kada je u pitanju imunoekspresija kaspaze 3, ATG7 i LC3. Kod hipotiroidnih pacova starih 4 dana smanjen je broj oogonija i oocita po nestu, primarnih i sekundarnih folikula, a povećan je broj primordijalnih folikula. Kod hipotiroidnih pacova starih 7 dana prisutan je veći broj primarnih, sekundarnih i preantralnih folikula. Alizarin Red bojenje i elektronska mikroskopija su ukazali na prisustvo prominentnog endoplazminog retikuluma kod hipotiroidnih pacova starih 4 dana.

Alternate abstract:

Thyroid hormones (TH) are essential for mammals’ development in the prenatal period. Previous studies have shown that subclinical hypothyroidism results in an increased rate of atresia in the pups ovaries in prepubertal and pubertal age. The impact of reduced concentration of TH on the early postnatal folliculogenesis and development of ovarian cancer has not been studied until now. The aim of the current thesis is to explain the influence of maternal hypothyroidism induced by 6-n-propyl-2-thiouracil (PTU) on folliculogenesis of rat offspring (n = 15) of at the rat at the day of birth (0. day) and 4 and 7 days old. The number and morphological characteristics of oogonia and oocytes of normal and altered morphology was determined using light and electron microscopy. These methods were applied to determine the number of germ cell nests, oogonia and oocytes within nest, primordial, primary, secondary and preantral follicles in the ovaries of 4 and 7 days old pups. The presence of glycogen and mucopolysaccharides in the ovary tissue of the pups at the 0. day was detected using PAS (Periodic Acid Shiff) histochemical staining and the presence of calcium by Alizarin Red staining in the ovary tissue of 4 and 7 days old pups. The volume density of nucleus, mitochondria and smooth endoplasmic reticulum in oocytes of primordial follicles was obtained in the ovaries 4 and 7 days old rats. Immunohistochemistry was used to detect the expression of PCNA (proliferating cell nuclear antigen), as a marker of proliferation, two markers of apoptosis (caspase-3 and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)) as well as two markers for autophagy (ATG7 LC3I / II). Hypothyroid rats at birth had reduced number of resting oogonia and oogonia undergoing mitosis, as well as the increased number of atypical oogonia/oocytes. Ultrastructural analyses showed the presence of degenerated cells in pachytene (Z-cell), oogonia/oocytes in autophagy, apoptosis and the combination of these two types of cell death in both groups of animals of this age. The most common types of atypical cells in hypothyroid rats at birth were oogonia/oocytes autophagy and Z-cell. In addition to the accelerated maturation morphologically unchanged oogonia/oocytes in this group of animals was the finding of the presence of prominent organelles in oocyte cytoplasm, followed with the increased expression of PCNA. Number of oogonia/oocytes labeled with TUNEL was lower in hypothyroid rats at birth, while there was no difference when it comes to expression of caspase-3, ATG7 and LC3.