In Vitro Mimicking of Estrous Cycle Stages in Porcine Oviduct Epithelium Cells: Estradiol and Progesterone Regulate Differentiation, Gene Expression, and Cellular Function

Biology of Reproduction (Impact Factor: 3.32). 07/2013; 89(3). DOI: 10.1095/biolreprod.113.108829
Source: PubMed


Throughout estrous cycle the oviduct epithelium undergoes dramatic morphological and functional changes. To elucidate cyclic cellular events and associated regulation mechanisms of estradiol (E2) and progesterone (P4), we mimicked estrous cycle stages in vitro using a culture system of primary porcine oviduct epithelium cells (POEC). Cells were polarized in an air/liquid interface, and then treated with E2 and P4 for physiological time periods: In experiment 1, high concentration of P4 with low concentration of E2 for 10 days resembled diestrus; in experiment 2, following the previous diestrus, sequential high E2 with low P4 for 2.5 days represented estrus. Histomorphometry and electron microscopy showed cyclic changes in cellular height, cell population and cilia density under influence of hormone stimulation. Trans-epithelial electrical resistance was high in simulated diestrus but reduced in estrus. Thus, E2 and P4 affect cellular polarity, transformation of ciliated and secretory cells, as well as electrical conductivity of oviduct epithelium. Simulation of diestrus led to significant decrease in expression of hormone receptors (PGR and ESR1) and other epithelial markers (MUC16, OVGP1 and HSP90B1), while sequential simulated estrus caused an increase in these markers. The hormonal regulation of some marker genes was clearly time dependent. Furthermore, POEC showed increased sperm binding capacity in simulated estrus. In this study we also present a novel approach based on the AndroVision software which can be routinely utilized as a parameter for ciliary activity and for the first time show fluid movement patterns along the epithelium lining in vitro.

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Available from: Jennifer Schoen, Mar 03, 2014
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    • "Controls (cells treated with ethanol solvent only) were included in the hormonal stimulation experiment. A total of 15 histomorphometric height measurements were taken for each sample following a recently published protocol (Chen et al. 2013a). "
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    ABSTRACT: Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphology and functionality. However, due to the optical properties of the filter materials and the cells' columnar shape, cell quality is hard to assess via light microscopy. In this study, we aim to evaluate transepithelial electrical resistance (TEER) measurement as a prognostic quality indicator for the cultivation of porcine oviduct epithelial cells (POEC). POEC were maintained in four different types of media for 3 and 6 w to achieve diverse culture qualities, and TEER was measured before processing samples for histology. Culture quality was scored using morphological criteria (presence of cilia, confluence and cell polarity). We furthermore analyzed the correlation between cellular height (as a measure of apical-basal polarization) and TEER in fully differentiated routine cultures (biological variation) and in cultures with altered cellular height due to hormonal stimulation. Fully differentiated cultures possessed a moderate TEER between 500 and 1100 Ω*cm(2). Only 5 % of cultures which exhibited TEER values in this defined range had poor quality. Sub-differentiated cultures showed either very low or excessively high TEER. We unveiled a highly significant (P < 0.0001) negative linear correlation between TEER and epithelial height in well-differentiated cultures (both routine and hormone stimulated group). This may point toward the interaction between tight junction assembly and epithelial apical-basal polarization. In conclusion, TEER is a straightforward quality indicator which could be routinely used to monitor the differentiation status of oviduct epithelial cells in vitro.
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    ABSTRACT: Oviducts play a critical role in gamete and embryo transport, as well as supporting early embryo development. Progesterone receptor (PGR) is a transcription factor highly expressed in oviductal cells, while its activating ligand, progesterone, surges to peak levels as ovulation approaches. Progesterone is known to regulate oviduct cilia beating and muscular contractions in vitro, but how PGR may mediate this in vivo is poorly understood. We used PGR null mice to identify genes potentially regulated by PGR in the oviducts during the periovulatory period. Histologically, oviducts from PGR null mice showed no gross structural or morphological defects compared to normal, littermates. However, microarray analysis of oviducts at 8 h post-hCG revealed over 1000 PGR-dependent genes. Ten genes were selected for validation using reverse-transcription polymerase chain reaction (RT-PCR) based on their potential roles in oocyte/embryo transport and support. Eight genes were confirmed to be down-regulated (Adamts1, Itga8, Edn3, Prlr, Ptgfr, Des, Myocd and Actg2) and one up-regulated (Agtr2) in PGR null oviducts. Expression of these genes was also assessed in oviducts of naturally cycling mice during ovulation, day 1 and day 4 of pregnancy. Adamts1, Itga8, Edn3, Prlr and Ptgfr were significantly up-regulated in oviducts at ovulation/mating. However, most genes showed basal levels of expression at other times. The exceptions were Prlr and Ptgfr which showed pulsatile increases on day 1 and/or day 4 of pregnancy. This is the first, comprehensive study to elucidate putative PGR-regulated genes in the oviduct and reveals key downstream targets potentially mediating oocyte and embryo transport.
    Physiological Genomics 06/2014; 46(16). DOI:10.1152/physiolgenomics.00044.2014 · 2.37 Impact Factor
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    ABSTRACT: In vitro models that resemble cell function in vivo are needed to understand oviduct physiology. This study aimed to assess cell functions and insulin effects on bovine oviductal epithelial cells (BOECs) cultured in an air–liquid interface. BOECs (n = 6) were grown in conditioned Ham's F12, DMEM or Ham's F12/DMEM with 10% fetal calf serum (FCS) for 3 weeks. After selecting the most suitable medium (Ham's F12), increasing insulin concentrations (1 ng/mL, 20 ng/mL and 5 μg/mL) were applied, and cell morphology and trans-epithelial electrical resistance (TEER; n = 4) were evaluated after 3 and 6 weeks. Keratin immunohistochemistry and mRNA expression of oviductal glycoprotein 1 (OVGP1) and progesterone receptor (PGR) were conducted (n = 4) to assess cell differentiation. BOECs grown without insulin supplementation or with 1 ng/mL of insulin displayed polarization and secretory activity. However, cells exhibited only 50% of the height of their in vivo counterparts. Cultures supplemented with 20 ng/mL insulin showed the highest quality, but the 5 μg/mL concentration induced massive growth. TEER correlated negatively with insulin concentration (r = −0.459; p = 0.009). OVGP1 and PGR transcripts were still detectable after 3 and 6 weeks. Cellular localization of keratins closely resembled that of BOECs in vivo. Cultures showed heterogeneous expression of PGR and OVGP1 in response to estradiol (10 pg/mL). In summary, BOECs grown for long term in an air–liquid interface expressed markers of cell differentiation. Additionally, insulin supplementation (20 ng/mL) improved the cell morphology in vitro.
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