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Analysis of the proteins and peptides isolated from incubation medium of early equine conceptuses - preliminary results
400
8th ISEET Abstracts / Journal of Equine Veterinary Science 32 (2012) 397-422
reLH as reported (Meyers-Brown et al., Anim Reprod Sci 2011). A total of 31 embryos were obtained from 11 mares in a 70 day period; of these, 21 embryos were used for ESC isolation. In order to isolate the ICM, blastocysts were processed by either immunosurgery (n ¼ 8) or microsurgery (n ¼ 13). The ICM was cultured on inactivated mouse embryonic fibroblasts (MEF) in control (n ¼ 9) or inhibited (n ¼ 11) medium. Control culture medium consisted of DMEM/F12, 15% FBS, 2 mM glutamine, 1 % nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.4 mg/ mL human LIF. The inhibited medium was supplemented with 3 mM GSK3 inhibitor (CHIR99021), 0.5 mM MEK inhibitor (PD0325901), and 2.5 mM TGF inhibitor (A83-01). No difference in initial cell line establishment was observed between immunosurgery and microsurgery ICM isolation (6/8 and 12/13, respectively) or between control and inhibited medium (8/9 and 10/11). The resulting cell lines grew in colonies of tightly packed cells with a high nuclear to cytoplasmic ratio. Some of the lines have been cultured for up to seven passages and have been successfully cryopreserved. Immunofluorescence analysis indicates that some cells expressed the pluripotency marker OCT4; however, all the cells were positive for the trophectoderm (TE) marker CDX2. To clarify the interpretation of these results we performed immunostaining of whole equine embryos at different stages of development. At the morula stage OCT4 was highly expressed in all cells and no CDX2 staining was observed. At day 7, expanded blastocysts expressed CDX2 clearly localized to the TE while OCT4 was present in both ICM and TE cells, although at higher intensity in the ICM. At Day 9, expanded blastocyst presented OCT4 expression exclusively localized to the ICM and CDX2 to the TE. In summary, the use of recombinant gonadotropins was effective at providing a large number of equine embryos for use in ESC isolation. Also, marker characterization of in vivo-derived embryos indicates that equine pluripotent cells should be positive for OCT4 and negative for CDX2, suggesting that cell lines derived in our study are of the TE lineage. Further approaches to isolate pure populations of ICM cells and maintain the undifferentiated characteristics of these cells in vitro are in progress. Acknowledgements Funding for this project was provided by the Center for Equine Health, UC Davis. Analysis of the proteins and peptides isolated from incubation medium of early equine conceptuses preliminary results Sven Budik 1, Katharina Nöbauer 2, Ebrahim Razzazi-Fazeli 2, and Christine Aurich 1 1 Centre for Artificial Insemination and Embryo Transfer, 2 Institute for Animal Nutrition, University for Veterinary Sciences, 1210 Vienna, Austria In the equine species, many aspects of the mechanism of maternal recognition of pregnancy are still unclear. Most importantly, the existence and nature of the embryonic signal involved in this event has not been resolved so far. In the present study we have therefore investigated proteins
and peptides secreted by the early equine conceptus. Cyclic Shetland pony mares (n ¼ 5) were inseminated with semen from fertile Shetland pony stallions (n ¼ 4). Embryos were collected on days 10, 12, 14 and 16 of pregnancy. Only intact embryos (n ¼ 9) were washed in Ringer Lactate and cultivated in 7 ml D-MEM with penicillin streptomycin without serum for either 6 or 24 hours. Intactness of the embryos was examined after the appropriate incubation time and the incubation medium was immediately frozen at -80 C. After storage the samples were thawed on ice and concentrated using ultrafiltration (AmiconÒ Ultra-4 Centrifugal Filter Units with 3kDa cutoff). Samples of the incubation medium were run on SDS-PAGE peptide gels (Biorad, Criterion Peptide Gel, 10–20%) and silver stained. No protein was detected in incubation medium of day 10 embryos. In day 12 and 14 embryos, the protein pattern was similar. Several bands were detected and some proteins could be identified: a middle intense band at about 12 kDa, a very strong band at 19 kDa, a very weak band at about 35 kDa, a weak double band at about 40 kDa and a weak single band at about 50 kDa. Analysis by MALDI- TOF/TOF mass spectrometry elucidated the nature of the 19 kDa band as uterocalin. Under bands are still under analysis. Preliminary results suggest that the equine embryo is able to deliver quickly peptides to the surrounding aqueous environment since the concentrations between the 5 and 24 hours-incubation groups did not differ. Furthermore, uterocalin is delivered very efficiently from the embryo to the aqueous environment showing that the equine embryonic capsule is able to store sufficient amounts of this protein. Spectrophotometric color analysis of the equine endometrium S. Neuhauser, and J. Handler Clinic for Horses, Faculty of Veterinary Medicine, Freie Universität Berlin, Germany Evaluation of the color of the equine endometrium during hysteroscopy is based on subjective classification, which can be influenced by various factors, such as surface of the object, illumination, position and angle of the observer and individual color vision. Objective endoscopic analysis of the endometrial color might be useful for the breeding soundness evaluation of recipient mares in embryo transfer programs. Because of this, we analyzed the color of the endometrium of equine uteri from the abattoir by use of an objective spectrophotometric color analysis system. In total, 23 equine uteri were collected and dissected to uncover the entire endometrium. Photographs of the endometrial surface were captured and the color analysis was performed with a spectrophotometer (CM-700dÔ, Konika Minolta) and the SpectraMagicÔNX-software (Konika Minolta) based on the three-dimensional L*a*b* color space (Commission Internationale de l'Eclairage 1976) defined by the three coordinates (L* – lightness-axis, a* – red/green-axis and b* – yellow/blue-axis). A total of 420 measurements (three for each corpus, left and right horn) were performed using the standard illuminant D65 (daylight including ultraviolet light, color temperature: 6504K) and the condition d/8. For statistical analysis, the jmp 6.0 software (SAS Institute Inc.) was used. Color measurements of the uteri ranged from 43.0
8th ISEET Abstracts / Journal of Equine Veterinary Science 32 (2012) 397-422
reLH as reported (Meyers-Brown et al., Anim Reprod Sci 2011). A total of 31 embryos were obtained from 11 mares in a 70 day period; of these, 21 embryos were used for ESC isolation. In order to isolate the ICM, blastocysts were processed by either immunosurgery (n ¼ 8) or microsurgery (n ¼ 13). The ICM was cultured on inactivated mouse embryonic fibroblasts (MEF) in control (n ¼ 9) or inhibited (n ¼ 11) medium. Control culture medium consisted of DMEM/F12, 15% FBS, 2 mM glutamine, 1 % nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.4 mg/ mL human LIF. The inhibited medium was supplemented with 3 mM GSK3 inhibitor (CHIR99021), 0.5 mM MEK inhibitor (PD0325901), and 2.5 mM TGF inhibitor (A83-01). No difference in initial cell line establishment was observed between immunosurgery and microsurgery ICM isolation (6/8 and 12/13, respectively) or between control and inhibited medium (8/9 and 10/11). The resulting cell lines grew in colonies of tightly packed cells with a high nuclear to cytoplasmic ratio. Some of the lines have been cultured for up to seven passages and have been successfully cryopreserved. Immunofluorescence analysis indicates that some cells expressed the pluripotency marker OCT4; however, all the cells were positive for the trophectoderm (TE) marker CDX2. To clarify the interpretation of these results we performed immunostaining of whole equine embryos at different stages of development. At the morula stage OCT4 was highly expressed in all cells and no CDX2 staining was observed. At day 7, expanded blastocysts expressed CDX2 clearly localized to the TE while OCT4 was present in both ICM and TE cells, although at higher intensity in the ICM. At Day 9, expanded blastocyst presented OCT4 expression exclusively localized to the ICM and CDX2 to the TE. In summary, the use of recombinant gonadotropins was effective at providing a large number of equine embryos for use in ESC isolation. Also, marker characterization of in vivo-derived embryos indicates that equine pluripotent cells should be positive for OCT4 and negative for CDX2, suggesting that cell lines derived in our study are of the TE lineage. Further approaches to isolate pure populations of ICM cells and maintain the undifferentiated characteristics of these cells in vitro are in progress. Acknowledgements Funding for this project was provided by the Center for Equine Health, UC Davis. Analysis of the proteins and peptides isolated from incubation medium of early equine conceptuses preliminary results Sven Budik 1, Katharina Nöbauer 2, Ebrahim Razzazi-Fazeli 2, and Christine Aurich 1 1 Centre for Artificial Insemination and Embryo Transfer, 2 Institute for Animal Nutrition, University for Veterinary Sciences, 1210 Vienna, Austria In the equine species, many aspects of the mechanism of maternal recognition of pregnancy are still unclear. Most importantly, the existence and nature of the embryonic signal involved in this event has not been resolved so far. In the present study we have therefore investigated proteins
and peptides secreted by the early equine conceptus. Cyclic Shetland pony mares (n ¼ 5) were inseminated with semen from fertile Shetland pony stallions (n ¼ 4). Embryos were collected on days 10, 12, 14 and 16 of pregnancy. Only intact embryos (n ¼ 9) were washed in Ringer Lactate and cultivated in 7 ml D-MEM with penicillin streptomycin without serum for either 6 or 24 hours. Intactness of the embryos was examined after the appropriate incubation time and the incubation medium was immediately frozen at -80 C. After storage the samples were thawed on ice and concentrated using ultrafiltration (AmiconÒ Ultra-4 Centrifugal Filter Units with 3kDa cutoff). Samples of the incubation medium were run on SDS-PAGE peptide gels (Biorad, Criterion Peptide Gel, 10–20%) and silver stained. No protein was detected in incubation medium of day 10 embryos. In day 12 and 14 embryos, the protein pattern was similar. Several bands were detected and some proteins could be identified: a middle intense band at about 12 kDa, a very strong band at 19 kDa, a very weak band at about 35 kDa, a weak double band at about 40 kDa and a weak single band at about 50 kDa. Analysis by MALDI- TOF/TOF mass spectrometry elucidated the nature of the 19 kDa band as uterocalin. Under bands are still under analysis. Preliminary results suggest that the equine embryo is able to deliver quickly peptides to the surrounding aqueous environment since the concentrations between the 5 and 24 hours-incubation groups did not differ. Furthermore, uterocalin is delivered very efficiently from the embryo to the aqueous environment showing that the equine embryonic capsule is able to store sufficient amounts of this protein. Spectrophotometric color analysis of the equine endometrium S. Neuhauser, and J. Handler Clinic for Horses, Faculty of Veterinary Medicine, Freie Universität Berlin, Germany Evaluation of the color of the equine endometrium during hysteroscopy is based on subjective classification, which can be influenced by various factors, such as surface of the object, illumination, position and angle of the observer and individual color vision. Objective endoscopic analysis of the endometrial color might be useful for the breeding soundness evaluation of recipient mares in embryo transfer programs. Because of this, we analyzed the color of the endometrium of equine uteri from the abattoir by use of an objective spectrophotometric color analysis system. In total, 23 equine uteri were collected and dissected to uncover the entire endometrium. Photographs of the endometrial surface were captured and the color analysis was performed with a spectrophotometer (CM-700dÔ, Konika Minolta) and the SpectraMagicÔNX-software (Konika Minolta) based on the three-dimensional L*a*b* color space (Commission Internationale de l'Eclairage 1976) defined by the three coordinates (L* – lightness-axis, a* – red/green-axis and b* – yellow/blue-axis). A total of 420 measurements (three for each corpus, left and right horn) were performed using the standard illuminant D65 (daylight including ultraviolet light, color temperature: 6504K) and the condition d/8. For statistical analysis, the jmp 6.0 software (SAS Institute Inc.) was used. Color measurements of the uteri ranged from 43.0