Hipofizno-ovarijalni sistem pacova od fetalnog do peripubertalnog perioda života nakon intrauterinog izlaganja deksametazonu

Abstract

Tokom fetalnog razvića organizam prolazi kroz “kritične periode”, tokom kojih se dešavaju intenzivne ćelijske deobe, razvijanje i sazrevanje tkiva, organa i organskih sistema. Sredina u kojoj fetus raste i razvija se u velikoj meri odreñuje kvalitet razvojnog procesa, a svaka promena uslova dovodi do čitavog niza poremećaja, koji mogu biti praćeni smanjenjem fetalnog rasta ili IUGR (eng. Intrauterine growth retardation). Posledice IUGR su dugotrajne i ispoljavaju se u vidu različitih metaboličkih i kardiovaskularnih oboljenja u kasnijem životu. Fetalno okruženje na taj način odreñuje adultni fenotip, a njihova povezanost proučava se u okviru koncepta programiranja. Najčešće korišćeni eksperimentalni pristupi u proučavanju programiranja i efekta IUGR su dijeta majke koja vodi pothranjenosti, izazivanje stresne reakcije kod majke ili primena antenatalne terapije glukokortikoidima. Svaki od ovih eksperimentalnih modela različitim mehanizmima dovodi do izlaganja fetusa povećanoj koncentraciji glukokortikoida, koji u velikoj meri mogu da poremete fiziološki tok razvojnog procesa. Cilj ove studije je bio da se ustanovi da li je izlaganje fetusa pacova od 16. do 18. dana gestacije sintetičkom glukokortikoidu deksametazonu programiralo razvoj i funkciju hipofizno-ovarijalnog sistema od fetalnog do peripubertalnog perioda života. Predmet istraživanja u ovoj doktorskoj disertaciji bili su fetusi i ženke pacova koje su od 16. do 18. dana gestacije izloženi deksametazonu. Gravidne ženke pacova su tri uzastopna dana (od 16. do 18. dana gestacije) subkutano tretirane deksametazonom (Dexamethasonis phosphat - Dx, Krka, p.o., Novo Mesto) rastvorenom u fiziološkom rastvoru (0.9% NaCl), u dozi od 1.0, 0.5 i 0.5 mg Dx/kg t.m./dan. Kontrolna grupa gravidnih ženki primala je u istom periodu, adekvatnu zapreminu fiziološkog rastvora. Potomci gravidnih ženki koje su primale deksametazon (Dx), odnosno fiziološki rastvor (K) žrtvovani su u fetalnom (19. i 21. fetalni dan), neonatalnom (5. dan), infantilnom (16. dan) i peripubertalnom (38. dan) periodu života. Za obeležavanje gonadotropnih (FSH i LH) ćelija hipofize korišćeno je imunocitohemijsko bojenje, dok su preseci jajnika bojeni histološkim bojenjem hematoksilin & eozin. Obojeni preseci hipofiza i jajnika stereološki i morfometrijski su analizirani, uz pomoć newCast stereološkog softverskog paketa. Dobijene su vrednosti volumena hipofize i jajnika, kao i apsolutni broj FSH i LH ćelija, njihov volumen, volumenska i numerička gustina. Koncentracija FSH i LH u cirkulaciji odreñena je biohemijskom RIA metodom. Pored volumena, u fetalnom jajniku odreñen je i apsolutni broj zdravih i degenerisanih germinativnih ćelija, a u jajniku ženki u postnatalnom periodu broj zdravih i atretičnih primordijalnih, primarnih, višeslojnih i antralnih folikula, kao i broj žutih tela. Dobijeni rezultati su statistički obrañeni. Intrauterino izlaganje deksametazonu izazvalo je IUGR (eng. intrauterine growth retardation) koje se manifestuje značajno smanjenom (p<0.05) telesnom masom kod fetusa starih 21 dan, a značajno smanjenje (p<0.05) telesne mase zabeleženo je i u postnatalnim periodima Volumen hipofize i adenohipoze značajno su smanjeni (p<0.05) u fetalnom, neonatalnom, infantilnom i peripubertalnom periodu života. Analizirani stereološki parametri su se po istom obrascu menjali i kod FSH i kod LH ćelija, kod ženki pacova intrauterino izloženih deksametazonu u odnosu na kontrolu. Tako je broj gonadotropnih ćelija značajno smanjen (p<0.05) od fetalnog, preko neonatalnog, infantilnog do peripubertalnog perioda života, a volumen je značajno smanjen (p<0.05) samo 19. fetalnog dana. Volumenska i numerička gustina gonadotropnih ćelija značajno su smanjene (p<0.05) u neonatalnom i infantilnom periodu života. Koncentracije FSH i LH su značajno smanjene (p<0.05) u neonatalnom, infantilnom i peripubertalnom periodu života kod ženki intrauterino izloženih deksametazonu u odnosu na kontrolu.. Nakon intrauterinog izlaganja deksametazonu volumen jajnika se značajno smanjio (p<0.05) u svim ispitivanim periodima života u odnosu na kontrolne vrednosti. Jajnik ženki pacova u fetalnom periodu nema definitivnu histološku organizaciju, tako da se u njima uočava prisustvo zdravih i degenerisanih germinativnih ćelija, i somatskih ćelija. Kvantifikacijom germinativnih ćelija, ustanovljeno je da je deksametazon u jajniku 19 dana starih fetusa uticao na značajno povećanje (p<0.05) broja degenerisanih na račun zdravih germinativnih ćelija, dok ukupan broj germinativnih ćelija nije promenjen u odnosu na kontrolu. U jajniku 21 dan starih fetusa, deksametazon je uticao na značajno smanjenje (p<0.05) ukupnog broja germinativnih ćelija, kao i na značajno smanjenje broja zdravih, a povećanje broja degenerisanih germinativnih ćelija (p<0.05). U jajniku 5 i 16 dana starih ženki pacova intrauterino izloženih deksametazonu prisutne su iste klase zdravih i atretičnih folikula kao i kod kontrolnih životinja, odnosno primordijalni, primarni i višeslojni folikuli, ali u značajno smanjenom broju (p<0.05). Za razliku od jajnika kontrolnih peripubertalnih ženki, u kojima su prisutna žuta tela, u jajnicima peripubertalnih ženki intrauterino izloženih deksametazonu još uvek ih nema. Broj zdravih primordijalnih i primarnih folikula je značajno smanjen (p<0.05), dok je broj analiziranih klasa zdravih i atretičnih višeslojnih i antralnih folikula značajno povećan (p<0.05) u odnosu na kontrolne vrednosti. Rezultati ove studije nedvosmisleno pokazuju da je reproduktivni potencijal ženki koje su tokom fetalnog razvoja bile izložene deksametazonu smanjen, a polno sazrevanje odloženo. S obzirom da je deksametazon uticao u ključnom momentu razvoja hipofize i jajnika u fetalnom periodu, promene koje je izazvao su trajne, i bitno odreñuju kvalitet reproduktivnog života ženki. Ovakav rezultat ima veliki značaj i primenu u humanoj medicini, s obzirom da su uslovi koji do njega mogu dovesti deo svakodnevnog života savremene žene i buduće majke.

During fetal development an organism undergoes “critical periods” of intensive cell division as well as the growth and maturation of tissues, organs and organ systems. The fetal environment principally determines the quality of the developmental process, while variation of conditions may lead to a range of disorders followed by intrauterine growth retardation (IUGR). The consequences of IUGR are long-lasting and manifest through various metabolic and cardiovascular issues in later life. Thus, the fetal environment prejudices the adult phenotype, while their intertwining is elaborated with the programming concept. The most widely exploited experimental approaches in the field of programming and IUGR effects are maternal diet leading to offspring malnutrition, provoking stress to mother and antenatal glucocorticoid therapy. Each of these experimental approaches via different mechanisms leads to fetal exposure to elevated glucocorticoid levels, which may disrupt to a great extent the physiology of the developmental process. The aim of this study was to establish whether the exposure of rat fetuses to synthetic glucocorticoid dexamethasone, during the period between days 16 and 18 of gestation, programmed the development and function of the pituitary-ovarian system, starting from the fetal to the peripubertal period of life. The subject of this doctoral thesis investigation were rat fetuses and females exposed to dexamethasone from the 16th to the 18th gestational day. During three consecutive days (16–18 days of gestation) pregnant rat females were subcutaneously treated with dexamethasone (Dexamethasonis phosphate – Dx, Krka, p.o., Novo Mesto) dissolved in saline (0.9% NaCl), in doses of 1.0, 0.5 and 0.5 mg Dx/kg b.w./day. During the same period a control group of pregnant females received an adequate volume of saline. The offspring of pregnant rat females which received dexamethasone (Dx) or saline (K) were sacrificed during the fetal (19th and 21st fetal day), neonatal (5th day), infantile (16th day) and peripubertal (38th day) period of life. Gonadotropic (FSH and LH) pituitary cells were immunocytochemically labeled, while the ovary sections were histologically stained with hematoxylin and eosin. Thus prepared pituitary and ovary sections were stereologically and morphometrically analyzed using the newCast stereological software package. The values of pituitary and ovary volume were obtained, as well as the absolute number of FSH and LH cells, their volume, volume density and numerical density. FSH and LH blood levels were biochemically (RIA) determined. Besides volume, the absolute numbers of healthy and degenerated cells in the fetal ovary were determined, while the numbers of healthy and atretic primordial, primary, secondary and antral follicles as well as corpora lutea were determined in the postnatal female ovaries. The obtained data were statistically processed. Intrauterine exposure to dexamethasone caused IUGR, manifested with significantly (p<0.05) decreased body weight of 21 days old fetuses, but a significant (p<0.05) decrease of the same parameter was also observed during the postnatal period. Pituitary and its adeno- fraction volumes were significantly (p<0.05) decreased in the fetal, neonatal, infantile and peripubertal period of life. Compared to the controls, the analyzed stereological parameters demonstrated the same pattern of changes in both FSH and LH cells in the female rats exposed in utero to dexamethasone. Thus, the number of gonadotropic cells was significantly (p<0.05) decreased starting from the fetal, during the neonatal, infantile to the peripubertal period of life, while their volume was significantly (p<0.05) decreased only at fetal day 19. The volume and numerical density of gonadotropic cells were significantly (p<0.05) decreased in neonatal and infantile periods of life. Compared to the controls, the FSH and LH blood levels were significantly (p<0.05) decreased in neonatal, infantile and peripubertal periods of life in the female rats exposed in utero to dexamethasone. After intrauterine exposure to dexamethasone, the ovary volume was significantly (p<0.05) decreased in all the examined periods of life, compared to the control values. Female rat ovary, in the fetal period of development, does not have a definitive histological organization; therefore the presence of healthy and degenerated germinative cells, as well as somatic cells can be observed. Quantification of germinative cells showed that, in the ovaries of 19 days old fetuses, dexamethasone caused a significant (p<0.05) increase in degenerated cells number at the expense of the healthy germinative cells pool, while the total number of germinative cells was not changed in comparison with the controls. In the ovaries of 21 days old fetuses, dexamethasone caused a significant (p<0.05) decrease of germinative cells total number, as well as a significant (p<0.05) decrease of healthy and increase of degenerated cells number. In the ovaries of 5 and 16 days old female rats exposed in utero to dexamethasone the same categories of healthy and atretic follicles were present as in the controls, i.e. primordial, primary and secondary follicles, but in a significantly (p<0.05) reduced number. Unlike the control, peripubertal female ovaries which possess corpora lutea are still missing in the ovaries of peripubertal females exposed in utero to dexamethasone. The number of healthy primordial and primary follicles was significantly (p<0.05) decreased, while the number of the analyzed categories of healthy and atretic secondary and antral follicles was significantly (p<0.05) increased, compared to the control values. The results of this study definitely indicate that the reproductive potential of females exposed in utero to dexamethasone is decreased, while their sexual maturation is delayed. Considering that dexamethasone realized its effects at the crucial moment of pituitary and ovary development during the fetal period, the changes caused are permanent, and determine substantially the quality of female reproductive life. Such a result is very important and is applicable in human medicine, since the elaborated conditions are entwined with the everyday life of the modern woman and future mother.