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Evaluation of reference genes for the bovine corpus luteum
46
reproductive biology 13s (2013) 22–64
of the sexual cycle, and their relationship to ovarian sex steroids. The stage of the estrous cycle in 30 Holstein bovine was assessed based on the gross and histological appearance of the ovaries and uterus, and on blood steroid hormone levels. Tissue samples taken from the uterus were fixed in 10% formaldehyde for routine histological processing. Positive membrane and cytoplasmic stainings of varying intensity were determined in the uterus in the follicular and luteal phases of the sexual cycle for erbB/HER receptors in luminal and glandular epithelial cells, connective tissue and smooth muscle cells and vascular endothelial and smooth muscle cells. It was demonstrated that in particular the apical and basal membranes of luminal epithelial cells and the apical membrane of glandular epithelial cells reacted with erbB1/HER1 and erbB2/HER2 during both the follicular and luteal phases. The reaction for erbB3/HER3 and erbB4/HER4 was stronger in the cytoplasm of luminal and glandular epithelial cells, but was of heterogeneous character. In both the follicular and luteal phases, the percentage and staining intensity of luminal and superficial glandular epithelial cells reacting positively with the receptors erbB1/HER1, erbB2/HER2, erbB3/HER3 and erbB4/HER4 were higher than those of deep glandular epithelial and connective tissue cells ( p < 0.05). In conclusion, it was demonstrated that the expression of the erbB/HER receptor family varied between different cell types in the bovine uterus during the follicular and luteal phases. http://dx.doi.org/10.1016/j.repbio.2013.01.097 P4.19 Variations in the progesterone secretion by developing corpora lutea influence the rhythm of follicular growth in bovine ovaries J. Schneebeli Summaprada, Switzerland Regarding the number of dominant follicles [DF], which emerge in the ovaries between consecutive ovulations, most bovine oestrous cycles can be called either 2- or 3-wave-cycles [2WC; 3WC]. Since the luteal phases following unsuccessful mating disproportionately often prove to be 3WC, it was suggested that incompletely balanced luteinization might impair early embryonic development and also alter the rhythm of follicular growth. To test the second part of this hypothesis, luteal function was comparatively analyzed during 122 oestrous cycles which differed in the number of DF-waves (daily blood sampling for progesterone-RIA; ovarian palpation every second day) in Swiss Brown dairy cattle. While an overall comparison of the progesterone-secretion in 2WC and 3WC did not reveal obvious differences, the starting point of the second DF-wave proved to be a relevant parameter. Cows whose second DF began ‘‘early’’, i.e. before day 14 [d14; d1 = oestrus], showed significantly higher median progesterone concentrations on d6 and d7 as compared with animals whose second DF-wave started later. When the discriminating limit for ‘‘early’’ versus ‘‘late’’ was set to d10, the difference became more distinct, and evident during a longer period. Inversely, setting the limit to a term later than d15 obliterated the differences. In summary, variations in the progesterone-secretion by developing corpora lutea appear to markedly influence the rhythm of ovarian follicular growth. The present study suggests the assumption that early ‘‘high’’ (above-average) progesterone-secretion, favoring a short interval between the first two DF-waves, might be a so far unnoticed subfertility factor which directly or indirectly impairs embryonic development. http://dx.doi.org/10.1016/j.repbio.2013.01.098 P4.20 Investigation of the expression of endothelial progenitor markers in the bovine ovary K. Schoen, J. Plendl, K. Dietze, S. Kaessmeyer Institute of Veterinary Anatomy, Department of Veterinary Medicine, Freie University of Berlin, Berlin, Germany Vasculogenesis refers to the formation of new blood vessels by differentiation of endothelial progenitor cells (EPC) to endothelial cells. In a previous study examining the co-expression of VEGF-R2 and CD34, EPC were detected by immunohistochemistry and RT-qPCR in the corpus luteum of the bovine ovary. It was shown that EPC quantity is related to the luteal stages. The highest quantities of EPC-markers were expressed during the developmental phase of the corpus luteum. The objective of this study was to investigate the expression of Estrogen-Receptor-1 (ESR-1) and EPC-markers. CD31 was used as a marker for mature endothelial cells. Ovaries were collected at the abattoir and macroscopically classified in the four luteal stages: development, mature, regression, and pregnancy. All primers were designed using Primer3 software. Reference genes that had been validated in a former study were used. Evaluation of gene expression was performed using the software qbasePlus (Biogazelle NV, Zwijnaarde, Belgium). In all stages of the bovine corpus luteum, expression of CD34, VEGF-R, ESR-1 and CD31 showed a distinct relationship. CD31 expression values were highest in the regression stage. http://dx.doi.org/10.1016/j.repbio.2013.01.099 P4.21 Evaluation of reference genes for the bovine corpus luteum K. Schoen 1, J. Plendl 1, C. Gabler 2, S. Kaessmeyer 1 Institute of Veterinary Anatomy Department of Veterinary Medicine, Freie University of Berlin, Berlin, Germany 2 Institute of Veterinary Biochemistry, Department of Veterinary Medicine, Freie University of Berlin, Berlin, Germany 1
In order to be able to compare gene expression in studies using quantitative Polymerase-Chain-Reaction (qPCR), data normalization is a crucial step. Therefore it is essential to identify suitable reference genes. Ideally, these reference genes have to be stably
reproductive biology 13s (2013) 22–64
47
expressed within the examined tissues, regardless of localization and experimental set up. The aim of the present study was to identify adequate reference genes for the bovine ovary to avoid errors by qPCR data normalization. To determine stably expressed reference genes for the bovine ovary, twelve candidate genes were chosen and analyzed using a qPCR SYBR-green assay. The evaluation of gene expression was performed in different luteal phases using the software qbasePlus The expression stability of specific candidate reference genes varied drastically between the different samples. The reference genes GAPDH and beta-actin, which are frequently used for normalization, revealed considerable expression variation and cannot be recommended unconditionally for studies on the bovine ovary. The most stably expressed reference genes in all samples were the members of the ribosomal protein genes. Of the many genes we tested only three displayed the optimal characteristics of adequate reference genes, i.e., RPS18, RPS9 and UBA52. http://dx.doi.org/10.1016/j.repbio.2013.01.100 P4.22 Expression of b-defensin in the equine endometrium S. Schöniger, H. Gräfe, H.A. Schoon Institute of Pathology, Faculty of Veterinary Medicine, University of Leipzig, Germany Defensins are molecules of the innate immune system. The aim of this study was to examine if b-defensins are components of the uterine immune defence in the mare. Endometrial samples from 15 mares were examined by histopathology for evaluation of endometrial health, by RT-PCR for detection of equine b-defensin-1 (EBD1) mRNA, and by immunohistochemistry to characterize the cell populations expressing b-defensins. PCR products from 5 mares were submitted for sequencing. In examined endometrial tissue samples, the glandular morphology was secretory in 6, proliferative in 7, proliferativesecretory in 1 and maldifferentiated in 1 mare. Mild or moderate endometriosis was diagnosed in 12 mares; mild, moderate or marked angiosclerosis in 12 mares; and mild or moderate endometritis in 6 mares. In the endometrium of all mares, EBD1 was detected by PCR, und b-defensins by immunohistochemistry. Sequencing confirmed the presence of EBD1. In all mares, bdefensin immunostaining was observed in the surface epithelium. Glandular epithelial cells were immunopositive in 11 mares, stromal cells in 2 mares and vascular myocytes in 7 mares. In glands, immunopositive cells were predominantly located in superficial portions. Epithelial cells and vascular myocytes showed mostly a cytoplasmic immunostaining, whereas a nuclear staining was observed in stromal cells. This study confirms the expression of b-defensins in the equine endometrium. The cytoplasmic labelling indicates synthesis and secretion of b-defensins by endometrial epithelial cells. A nuclear staining suggests that b-defensins may act as transcription factors. It appears likely that also in mares b-defensins are important factors for maintaining endometrial health and fertility; the regulation of endometrial b-defensin expression has to be investigated in further studies. http://dx.doi.org/10.1016/j.repbio.2013.01.101
P4.23 Effect of cytokines on PGIS mRNA expression and PGI2 release from luminal epithelial cells of the porcine endometrium M. Szymańska, E. Morawska-Pucińska, A. Blitek Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland Prostacyclin (PGI2) is synthesized and secreted by the porcine endometrium and increased concentration of PGI2 was observed in pregnant animals beginning from day 12. However, there is little information concerning factors that may regulate PGI2 synthesis in this tissue. Cytokines and their receptors are present in the uterus. Thus, we examined the effect of selected cytokines on the expression of PGI2 synthase (PGIS) mRNA and the release of 6-keto PGF1a (PGI2 metabolite) by luminal epithelial (LE) cells of the endometrium collected from day 12 cyclic gilts. LE cells were cultured in collagen-coated 6-well plates in Medium 199 containing 5% newborn calf serum. After reaching 80% confluence the cells were treated with IL-6, LIF, IL-1b, TNFa and IFNg (all at the concentration of 10 ng/ml) in the presence of arachidonic acid. The medium for EIA of 6-keto PGF1a was collected after 3, 6 and 24 h of incubation. LE cells were collected after 6 and 24 h to determine PGIS mRNA expression using real-time PCR. Treatment of LE cells with cytokines had no effect on 6-keto PGF1a secretion after 3 h. Incubation of LE for 6 h resulted in increased concentration of PGI2 metabolite in medium in the presence of IL-1b and TNFa ( p < 0.01 and p < 0.001; respectively, compared to control). IL-6 and IFNg decreased the level of PGI2 in medium after 24 h ( p < 0.05 and p < 0.01; respectively). None of the studied cytokines affected PGIS mRNA expression in LE cells. In conclusion, cytokines may regulate the secretion but not synthesis of PGI2 in LE cells of the porcine endometrium. Supported by NSC (2011/01/B/NZ9/07069). http://dx.doi.org/10.1016/j.repbio.2013.01.102
P4.24 Effect of chitosan on 17b-estradiol secretion in C57/BL/6J mice exposed to PCBs A. Tomza-Marciniak, T. Stankiewicz, B. Pilarczyk, B. Błaszczyk, J. Kuba, D. Hendzel, J. Udała Department of Animal Reproduction Biotechnology and Environmental Hygiene, West Pomeranian University of Technology in Szczecin, Poland The aim of this study was to evaluate an effect of chitosan on negative polychlorinated biphenyls' (PCBs) influence on 17bestradiol secretion in mice.
reproductive biology 13s (2013) 22–64
of the sexual cycle, and their relationship to ovarian sex steroids. The stage of the estrous cycle in 30 Holstein bovine was assessed based on the gross and histological appearance of the ovaries and uterus, and on blood steroid hormone levels. Tissue samples taken from the uterus were fixed in 10% formaldehyde for routine histological processing. Positive membrane and cytoplasmic stainings of varying intensity were determined in the uterus in the follicular and luteal phases of the sexual cycle for erbB/HER receptors in luminal and glandular epithelial cells, connective tissue and smooth muscle cells and vascular endothelial and smooth muscle cells. It was demonstrated that in particular the apical and basal membranes of luminal epithelial cells and the apical membrane of glandular epithelial cells reacted with erbB1/HER1 and erbB2/HER2 during both the follicular and luteal phases. The reaction for erbB3/HER3 and erbB4/HER4 was stronger in the cytoplasm of luminal and glandular epithelial cells, but was of heterogeneous character. In both the follicular and luteal phases, the percentage and staining intensity of luminal and superficial glandular epithelial cells reacting positively with the receptors erbB1/HER1, erbB2/HER2, erbB3/HER3 and erbB4/HER4 were higher than those of deep glandular epithelial and connective tissue cells ( p < 0.05). In conclusion, it was demonstrated that the expression of the erbB/HER receptor family varied between different cell types in the bovine uterus during the follicular and luteal phases. http://dx.doi.org/10.1016/j.repbio.2013.01.097 P4.19 Variations in the progesterone secretion by developing corpora lutea influence the rhythm of follicular growth in bovine ovaries J. Schneebeli Summaprada, Switzerland Regarding the number of dominant follicles [DF], which emerge in the ovaries between consecutive ovulations, most bovine oestrous cycles can be called either 2- or 3-wave-cycles [2WC; 3WC]. Since the luteal phases following unsuccessful mating disproportionately often prove to be 3WC, it was suggested that incompletely balanced luteinization might impair early embryonic development and also alter the rhythm of follicular growth. To test the second part of this hypothesis, luteal function was comparatively analyzed during 122 oestrous cycles which differed in the number of DF-waves (daily blood sampling for progesterone-RIA; ovarian palpation every second day) in Swiss Brown dairy cattle. While an overall comparison of the progesterone-secretion in 2WC and 3WC did not reveal obvious differences, the starting point of the second DF-wave proved to be a relevant parameter. Cows whose second DF began ‘‘early’’, i.e. before day 14 [d14; d1 = oestrus], showed significantly higher median progesterone concentrations on d6 and d7 as compared with animals whose second DF-wave started later. When the discriminating limit for ‘‘early’’ versus ‘‘late’’ was set to d10, the difference became more distinct, and evident during a longer period. Inversely, setting the limit to a term later than d15 obliterated the differences. In summary, variations in the progesterone-secretion by developing corpora lutea appear to markedly influence the rhythm of ovarian follicular growth. The present study suggests the assumption that early ‘‘high’’ (above-average) progesterone-secretion, favoring a short interval between the first two DF-waves, might be a so far unnoticed subfertility factor which directly or indirectly impairs embryonic development. http://dx.doi.org/10.1016/j.repbio.2013.01.098 P4.20 Investigation of the expression of endothelial progenitor markers in the bovine ovary K. Schoen, J. Plendl, K. Dietze, S. Kaessmeyer Institute of Veterinary Anatomy, Department of Veterinary Medicine, Freie University of Berlin, Berlin, Germany Vasculogenesis refers to the formation of new blood vessels by differentiation of endothelial progenitor cells (EPC) to endothelial cells. In a previous study examining the co-expression of VEGF-R2 and CD34, EPC were detected by immunohistochemistry and RT-qPCR in the corpus luteum of the bovine ovary. It was shown that EPC quantity is related to the luteal stages. The highest quantities of EPC-markers were expressed during the developmental phase of the corpus luteum. The objective of this study was to investigate the expression of Estrogen-Receptor-1 (ESR-1) and EPC-markers. CD31 was used as a marker for mature endothelial cells. Ovaries were collected at the abattoir and macroscopically classified in the four luteal stages: development, mature, regression, and pregnancy. All primers were designed using Primer3 software. Reference genes that had been validated in a former study were used. Evaluation of gene expression was performed using the software qbasePlus (Biogazelle NV, Zwijnaarde, Belgium). In all stages of the bovine corpus luteum, expression of CD34, VEGF-R, ESR-1 and CD31 showed a distinct relationship. CD31 expression values were highest in the regression stage. http://dx.doi.org/10.1016/j.repbio.2013.01.099 P4.21 Evaluation of reference genes for the bovine corpus luteum K. Schoen 1, J. Plendl 1, C. Gabler 2, S. Kaessmeyer 1 Institute of Veterinary Anatomy Department of Veterinary Medicine, Freie University of Berlin, Berlin, Germany 2 Institute of Veterinary Biochemistry, Department of Veterinary Medicine, Freie University of Berlin, Berlin, Germany 1
In order to be able to compare gene expression in studies using quantitative Polymerase-Chain-Reaction (qPCR), data normalization is a crucial step. Therefore it is essential to identify suitable reference genes. Ideally, these reference genes have to be stably
reproductive biology 13s (2013) 22–64
47
expressed within the examined tissues, regardless of localization and experimental set up. The aim of the present study was to identify adequate reference genes for the bovine ovary to avoid errors by qPCR data normalization. To determine stably expressed reference genes for the bovine ovary, twelve candidate genes were chosen and analyzed using a qPCR SYBR-green assay. The evaluation of gene expression was performed in different luteal phases using the software qbasePlus The expression stability of specific candidate reference genes varied drastically between the different samples. The reference genes GAPDH and beta-actin, which are frequently used for normalization, revealed considerable expression variation and cannot be recommended unconditionally for studies on the bovine ovary. The most stably expressed reference genes in all samples were the members of the ribosomal protein genes. Of the many genes we tested only three displayed the optimal characteristics of adequate reference genes, i.e., RPS18, RPS9 and UBA52. http://dx.doi.org/10.1016/j.repbio.2013.01.100 P4.22 Expression of b-defensin in the equine endometrium S. Schöniger, H. Gräfe, H.A. Schoon Institute of Pathology, Faculty of Veterinary Medicine, University of Leipzig, Germany Defensins are molecules of the innate immune system. The aim of this study was to examine if b-defensins are components of the uterine immune defence in the mare. Endometrial samples from 15 mares were examined by histopathology for evaluation of endometrial health, by RT-PCR for detection of equine b-defensin-1 (EBD1) mRNA, and by immunohistochemistry to characterize the cell populations expressing b-defensins. PCR products from 5 mares were submitted for sequencing. In examined endometrial tissue samples, the glandular morphology was secretory in 6, proliferative in 7, proliferativesecretory in 1 and maldifferentiated in 1 mare. Mild or moderate endometriosis was diagnosed in 12 mares; mild, moderate or marked angiosclerosis in 12 mares; and mild or moderate endometritis in 6 mares. In the endometrium of all mares, EBD1 was detected by PCR, und b-defensins by immunohistochemistry. Sequencing confirmed the presence of EBD1. In all mares, bdefensin immunostaining was observed in the surface epithelium. Glandular epithelial cells were immunopositive in 11 mares, stromal cells in 2 mares and vascular myocytes in 7 mares. In glands, immunopositive cells were predominantly located in superficial portions. Epithelial cells and vascular myocytes showed mostly a cytoplasmic immunostaining, whereas a nuclear staining was observed in stromal cells. This study confirms the expression of b-defensins in the equine endometrium. The cytoplasmic labelling indicates synthesis and secretion of b-defensins by endometrial epithelial cells. A nuclear staining suggests that b-defensins may act as transcription factors. It appears likely that also in mares b-defensins are important factors for maintaining endometrial health and fertility; the regulation of endometrial b-defensin expression has to be investigated in further studies. http://dx.doi.org/10.1016/j.repbio.2013.01.101
P4.23 Effect of cytokines on PGIS mRNA expression and PGI2 release from luminal epithelial cells of the porcine endometrium M. Szymańska, E. Morawska-Pucińska, A. Blitek Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland Prostacyclin (PGI2) is synthesized and secreted by the porcine endometrium and increased concentration of PGI2 was observed in pregnant animals beginning from day 12. However, there is little information concerning factors that may regulate PGI2 synthesis in this tissue. Cytokines and their receptors are present in the uterus. Thus, we examined the effect of selected cytokines on the expression of PGI2 synthase (PGIS) mRNA and the release of 6-keto PGF1a (PGI2 metabolite) by luminal epithelial (LE) cells of the endometrium collected from day 12 cyclic gilts. LE cells were cultured in collagen-coated 6-well plates in Medium 199 containing 5% newborn calf serum. After reaching 80% confluence the cells were treated with IL-6, LIF, IL-1b, TNFa and IFNg (all at the concentration of 10 ng/ml) in the presence of arachidonic acid. The medium for EIA of 6-keto PGF1a was collected after 3, 6 and 24 h of incubation. LE cells were collected after 6 and 24 h to determine PGIS mRNA expression using real-time PCR. Treatment of LE cells with cytokines had no effect on 6-keto PGF1a secretion after 3 h. Incubation of LE for 6 h resulted in increased concentration of PGI2 metabolite in medium in the presence of IL-1b and TNFa ( p < 0.01 and p < 0.001; respectively, compared to control). IL-6 and IFNg decreased the level of PGI2 in medium after 24 h ( p < 0.05 and p < 0.01; respectively). None of the studied cytokines affected PGIS mRNA expression in LE cells. In conclusion, cytokines may regulate the secretion but not synthesis of PGI2 in LE cells of the porcine endometrium. Supported by NSC (2011/01/B/NZ9/07069). http://dx.doi.org/10.1016/j.repbio.2013.01.102
P4.24 Effect of chitosan on 17b-estradiol secretion in C57/BL/6J mice exposed to PCBs A. Tomza-Marciniak, T. Stankiewicz, B. Pilarczyk, B. Błaszczyk, J. Kuba, D. Hendzel, J. Udała Department of Animal Reproduction Biotechnology and Environmental Hygiene, West Pomeranian University of Technology in Szczecin, Poland The aim of this study was to evaluate an effect of chitosan on negative polychlorinated biphenyls' (PCBs) influence on 17bestradiol secretion in mice.