Prenatal exposure to antiandrogen flutamide affects androgen receptor (AR) expression in postnatal ovarian development in pig

The following study was undertaken to localize androgen receptors (ARs) in various structures of the porcine ovary after prenatal exposure to antiandrogen flutamide. In wet. treatment by antiandrogens may have adverse effects on reproductive function in immature and adult animals. Flutamide was injected into pregnant swines between days 20 and 28 (GD20) or 80 to 88 (GD80) of gestation. The ovaries were collected from treated animals and from control ones (non-treated) at two different points of development: from immature and adult pigs. Immunoexpression of AR was determined for preantral and antral follicles and for stroma cells. Immunostaining showed that AR expression in immature animals was unaffected in the primary follicles, while in the preantral and antral follicles the AR level fluctuated depending on day of treatment as well as on analyzed tissue. In adult animals, the immunoexpression of AR slightly decreased in antral follicles independently on the day of flutamide treatment. Therefore. AR expression in postnatal life may be affected by in utero exposure to antiandrogen flutamide

The impact of antiandrogen flutamide on the hypoxia inducible factor 1a and vascular endothelial growth factor A gene and protein expression in the pig placenta during late pregnancy

Introduction. In contrast to estradiol action, little is known about androgen signaling in placental development. The purpose of this study was to evaluate the impact of diminished androgen action on hypoxia inducible factor 1a (HIF-1a) and vascular endothelial growth factor A (VEGFA) protein expression as well as their mRNAs in the structures of fetal and maternal parts of porcine placenta during late pregnancy. Material and methods. Pregnant pigs were injected daily with antiandrogen flutamide, at a dose of 50 mg/kg body weight at different stages of pregnancy: between gestational days 83–89 (90 dpc) and 101–107 (108 dpc). Control groups (90 dpc or 108 dpc) were treated with vehicle (corn oil). One day after the last injection animals were sacrificed and tissues were collected. Tissue samples were frozen for mRNA isolation or fixed for immu­nohistochemistry (IHC). The expression of HIF-1a and VEGFA were investigated by real-time PCR and IHC. Results. Flutamide treatment caused changes in both HIF-1a and VEGFA mRNA levels only in the placentas of the 90 dpc group. Relative optical density analysis showed decreased HIF-1a and increased VEGFA protein expression in the placentas obtained from flutamide-treated 108 dpc group while no differences were observed in the 90 dpc group. Conclusions. Experimentally induced androgen deficiency in pigs deregulates the expression of some genes important for placental blood circulation. We suggest that androgens are involved in the control of expression of HIF-1a and VEGFA in porcine placenta during late pregnancy

Influence of antiandrogen flutamide on gene expression with the pig plaeenta

W czasie ciąży powstają dwa unikatowe, tymczasowe organy - łożysko oraz sznur pępowinowy. Łożysko bierze udział w aktywnej wymianie składników odżywczych, gazów oddechowych oraz zbędnych produktów przemiany materii, które są transportowane poprzez naczynia sznura pępowinowego pomiędzy organizmem matki, a organizmami płodów. Od prawidłowego ukształtowania oraz funkcjonowania organów tymczasowych zależy efektywność wymiany w obrębie jednostki matczyno-płodowej, a tym samym sukces całej ciąży. W czasie powstawania organów tymczasowych ważną rolę odgrywają m.in. układ czynnika VEGF - odpowiedzialny za proces waskulogenezy i angiogenezy oraz powstawanie złącz szczelionowych, umożliwiających transport cząsteczek pomiędzy komórkami. Dane dotyczące roli hormonów steroidowych w kontroli powyższych procesów są słabo poznane. Dlatego też, celem przeprowadzonych badań było określenie wpływu androgenów na ekspresję genów recptora androgenowego (AR), koneksyny 43 (Cx43), czynnika wzrostu śródbłonka naczyń krwionośnych (VEGFa) oraz jego dwóch receptorów: Flt-1 i KDR, w jednostce matczyno-płodowej, macicach pomiędzy jajami płodowymi oraz sznurach pępowinowych świni. W badaniach zastosowano model badawczy z wykorzystaniem flutamidu (niesteroidowego antyandrogenu, bloker AR), który podawano ciężarnym świniom w różnych ważnych dla rozwoju organów tymczasowych okresach ciąży tj. od 43-49 dpc (n=2), 83-89 dpc (n=2), 101-107 dpc (n=2). Materiał do badań pobrano w 50, 90 i 108 dpc. W celu oceny histologicznej pobranych tkanek przeprowadzono analizę morfologiczną. Aby ustalić poziomy fizjologiczne ekspresji badanych białek oraz zmiany wywołane niedoborem androgenów przeprowadzono analizę Real time PCR, Western blot oraz analizę immunohistochemiczną. Ocena histologiczna badanych tkanek nie wykazała zmian morfologicznych wywołanych działaniem flutamidu w obrębie komórek budujących poszczególne komponenty badanych organów tymczasowych oraz fragmentów macic pobranych pomiędzy jajami płodowymi świni. Flutamid wywołał zmiany na poziomie molekularnym, zaburzając ekspresję badanych genów zarówno na poziomie mRNA jak i białka na różnych badanych etapach ciąży. Zaobserwowano zróżnicowaną odpowiedź komórek na wywołany ograniczony dostęp androgenów, który powodował wzrost lub spadek ekspresji badanych genów. Sposób zmiany ekspresji nie był skorelowany z konkretnym typem badanych komórek ani etapem ciąży. Na podstawie uzyskanych wyników nie można wykluczyć roli androgenów w regulacji ekspresji badanych genów biorących udział w procesie angiogenezy i tworzeniu połączeń międzykomórkowych, co w przypadku funkcji jakie pełnią organy tymczasowe jest kwestią istotną dla prawidłowej ich fizjologii oraz sukcesu ciąży.Two unique and temporary organs are formed during pregnancy: a placenta and an umbilical cord. The placenta takes part in active nutrients, respiratory gases and waste products exchange, which are transported by the umbilical cord blood vessels between the mother's and fetuse's body. Successful pregnancy and efficient exchange within the materno-fetal surface depends on the proper formation and functioning of these temporary organs. During their formation a Vascular Endothelial Growth Factor (VEGFa) and its receptors as well as Connexin 43 (Cx43) protein play an important role. The VEGFa takes part in the new blood vessels formation (vasculogenesis) and its expansion (angiogenesis), while the Cx43 formes the gap junctions which allows molecules exchange between adjacent cells. Role of androgens in the control of these processes is still poorly understood. Therefore, the aim of this thesis was to determine the influence of androgens on the androgen receptor (AR), Cx43, VEGFa, Vascular Endothelial Growth Factor type 1 (Flt-1), Vascular Endothelial Growth Factor type 2 (KDR) genes expression within: the materno-fetal surface, the uteri between fetuses and the umbilical cords. In the present study the flutamide - a nonsteroid antiandrogen that blocks ARs, was used. The flutamide was administreted as an injection to pregnant pigs in times important for development of the temporary organs i.e. 43-49 day post coitum (dpc), 83-89 dpc and 101-107 dpc. The material for investigation was collected the day after injections i.e. 50, 90, 108 dpc. To estimate the morphological changes standard histological analysis was carried out. To determine physiological changes in gene expression induced by androgens deficiency the Real time PCR, the Western blot and the immunohistochemical analysis were used. Flutamide did not induce the morphological changes within investigated cells building temporary organs and the uteri between fetuses, but inducedchanges at a molecular lavel. Up and down regulation of the AR, Cx43, VEGFa, Flt-1, KDR expression was observed at mRNA and protein level however these changes were not correlated with the cell types and examined time of pregnancy. Based on the obtained results, the importance of androgens role in the regulation of investigated genes expression and thereby regulation of angiogenesis, vasculogenesis and cell communication cannot be excluded

Androgen deficiency during mid-and late pregnancy alters progesterone production and metabolism in the porcine corpus luteum

We determined whether androgen deficiency induced by flutamide treatment during mid- and late pregnancy affects the functions of the porcine corpus luteum (CL). Pregnant gilts were injected with flutamide between days 43 and 49 (gestation day [GD] 50F), days 83 and 89 (GD90F), or days 101 and 107 (GD108F) of gestation. Antiandrogen treatment increased the luteal progesterone concentration in the GD50F group and decreased progesterone content in the GD90F and GD108F groups. Luteal levels of side-chain cleavage cytochrome P450 (CYP11A1) mRNA and protein were significantly downregulated in the GD90F and GD108F groups as compared with the respective controls. The 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (HSD3B) mRNA and protein expression were significantly reduced only in the GD108F group as compared with the control. Decreased luteal 20α-hydroxysteroid dehydrogenase (AKR1C1) mRNA and protein levels were observed in the GD50F group. Thus, androgen deficiency during pregnancy in pigs led to CL dysfunction that is marked by decreased progesterone production. Furthermore, exposure to flutamide during late pregnancy downregulated steroidogenic enzymes (CYP11A1 and HSD3B) in pigs. We conclude that androgens are important regulators of CL function during pregnancy