ABSTRACT
Androgen deficiency during prenatal development may affect the expression of genes involved in the folliculogenesis regulation. In order to study the effect of antiandrogen on fetal ovarian development, pregnant gilts were injected with flutamide (for 7 days, 50 mg/kg bodyweight per day) or corn oil (control groups) starting on gestation days 43 (GD50), 83 (GD90), or 101 (GD108). The obtained fetal ovaries were fixed for histology and immunohistochemistry or frozen for real-time PCR. Morphological evaluation, TUNEL assay, and expression of selected factors (Ki-67, GATA binding transcription factor 4 (GATA4), E-Cadherin and tumor necrosis factor a (TNFa)) were performed. On GD90 and GD108, ovaries following flutamide administration showed a higher number of egg nests and lower number off ollicles than those in respective control groups. An increased mRNA and protein expression of Ki-67 was observed in flutamide-treated groups compared with controls on GD50 and GD108 but decreased expression was found on GD90. In comparison to control groups a higher percentage of TUNEL-positive cells was shown after flutamide exposure on GD50 and GD90 and a lower percentage of apoptotic cells was observed on GD108. These data were consistent with changes in TNF (TNFa) mRNA expression, which increased on GD90 and decreased on GD108. E-cadherin mRNA and protein expression was upregulated on GD50 and downregulated on GD90 and GD108. In conclusion diminished androgen action in porcine fetal ovaries during mid- and late gestation leads to changes in the expression of genes crucial for follicle formation. Consequently, delayed folliculogenesis was observed on GD90 and GD108. It seems however that androgens exhibit diverse biological effects depending on the gestational period.
Subject(s)
Androgen Antagonists/pharmacology , Fetus/drug effects , Flutamide/pharmacology , Ovary/drug effects , Androgen Antagonists/administration & dosage , Animals , Apoptosis/drug effects , Cadherins/genetics , Cadherins/metabolism , Cell Proliferation/drug effects , Female , Fetus/metabolism , Fetus/pathology , Flutamide/administration & dosage , GATA4 Transcription Factor/genetics , GATA4 Transcription Factor/metabolism , Gene Expression Regulation, Developmental/drug effects , Gestational Age , Immunohistochemistry , In Situ Nick-End Labeling , Injections, Subcutaneous , Ki-67 Antigen/genetics , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism , Ovarian Follicle/pathology , Ovary/metabolism , Ovary/pathology , Pregnancy , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Swine , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The aim of this study was to determine the immunolocalization and expression of the androgen receptor (AR) in the pig placenta and umbilical cord during pregnancy following exposure to flutamide, a non-steroidal antiandrogen, at its various stages. Pregnant pigs were injected with flutamide at a daily dose of 50mg/kg body weight at different stages of pregnancy: from day 83-89 (n=2); from day 101-107 (n=2). They were sacrificed and tissues collected one day after the last injection. Control animals, two for each experimental point, were injected only with the vehicle (corn oil). Collected tissue samples were fixed for immunohistochemistry or frozen for protein isolation. AR protein was detected in the nucleus of trophoblast cells forming the structure of ridges and in maternal endothelial cells, which are involved in the placental barrier formation. It was also localized in the nuclei of cells forming umbilical cord components: allantoic duct epithelium, amniotic epithelium, Wharton's jelly and the muscular layer of the umbilical cord vein and arteries. Relative optical density analysis showed increased expression in the material derived from animals treated with flutamide. The presence of AR in the placental barrier and in the umbilical cord components suggests a role of androgen in those temporary organs. Flutamide could impact on the levels of the AR protein in the reproductive tracts during pregnancy in sows.
Subject(s)
Flutamide/pharmacology , Placenta/metabolism , Receptors, Androgen/metabolism , Umbilical Cord/metabolism , Animals , Blotting, Western , Female , Gene Expression Regulation , Immunohistochemistry , Placenta/chemistry , Placenta/cytology , Pregnancy , Receptors, Androgen/analysis , Receptors, Androgen/genetics , Swine , Umbilical Cord/chemistry , Umbilical Cord/cytologyABSTRACT
The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T+2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6-8mm in diameter) were cultured for 48 h. Experimental cultures were carried out with the addition of T (10(-7)M), 2-Hf (1.7×10(-4)M) or both T and 2-Hf for the last 24 h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6-8mm in diameter) were cultured for 24 h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P(4)) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P(4) secretion, but simultaneous addition of 2-Hf and T increased P(4) secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.