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Deborah J. Guest - One of the best experts on this subject based on the ideXlab platform.
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three dimensional culture and transforming growth factor beta3 synergistically promote tenogenic differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2014Co-Authors: Tom Barsby, Emma P Bavin, Deborah J. GuestAbstract:The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of Equine Embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared...
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three dimensional culture and transforming growth factor beta3 synergistically promote tenogenic differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2014Co-Authors: Tom Barsby, Emma P Bavin, Deborah J. GuestAbstract:The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of Equine Embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared with tenogenic induction in monolayer ESC cultures. The addition of TGF-β3 to the 3D cultures further up-regulates the expression of these genes and also induces the expression of mature tenocyte markers Tenomodulin and Thrombospondin-4. Our results show that when ESCs are exposed to the intrinsic forces exerted by a 3D culture environment, they express tendon-associated genes and proteins which are indicative of tenocyte lineage differentiation and that this effect is synergistically enhanced and accelerated by the addition of TGF-β3.
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transforming growth factor beta3 promotes tendon differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2013Co-Authors: Tom Barsby, Deborah J. GuestAbstract:Tendon injuries occur frequently in horses and have a poor capacity to regenerate, which leads to high re-injury rates. Equine Embryo-derived stem cells (ESCs) survive in high numbers in the injured horse tendon and we hypothesized that they differentiate into tenocytes in vivo. Immunocytochemistry revealed that in the injured horse tendon ESCs express the tendon progenitor marker scleraxis and that there is a local upregulation of the transforming growth factor-β (TGF-β) at the injury site. The aim of this study was to determine if TGF-β signaling was able to drive tenocyte differentiation by ESCs. Exposure of differentiating ESCs to TGF-β in vitro produced an upregulation of scleraxis at the gene and protein level with the greatest effect being produced in the presence of TGF-β3. TGF-β3 treatment of differentiating ESCs also promotes a significant upregulation of other tendon-associated genes and proteins suggesting it can promote ESC differentiation into tenocytes. Our results demonstrate that Equine E...
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transforming growth factor beta3 promotes tendon differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2013Co-Authors: Tom Barsby, Deborah J. GuestAbstract:Tendon injuries occur frequently in horses and have a poor capacity to regenerate, which leads to high re-injury rates. Equine Embryo-derived stem cells (ESCs) survive in high numbers in the injured horse tendon and we hypothesized that they differentiate into tenocytes in vivo. Immunocytochemistry revealed that in the injured horse tendon ESCs express the tendon progenitor marker scleraxis and that there is a local upregulation of the transforming growth factor-β (TGF-β) at the injury site. The aim of this study was to determine if TGF-β signaling was able to drive tenocyte differentiation by ESCs. Exposure of differentiating ESCs to TGF-β in vitro produced an upregulation of scleraxis at the gene and protein level with the greatest effect being produced in the presence of TGF-β3. TGF-β3 treatment of differentiating ESCs also promotes a significant upregulation of other tendon-associated genes and proteins suggesting it can promote ESC differentiation into tenocytes. Our results demonstrate that Equine ESCs can differentiate into a therapeutically relevant cell type and that TGF-β driven differentiation of ESCs may provide a model to study tendon development and better understand the transcriptional networks that are involved in Equine tendon cell differentiation from the early Embryonic stages.
Tom Barsby - One of the best experts on this subject based on the ideXlab platform.
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three dimensional culture and transforming growth factor beta3 synergistically promote tenogenic differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2014Co-Authors: Tom Barsby, Emma P Bavin, Deborah J. GuestAbstract:The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of Equine Embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared...
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three dimensional culture and transforming growth factor beta3 synergistically promote tenogenic differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2014Co-Authors: Tom Barsby, Emma P Bavin, Deborah J. GuestAbstract:The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of Equine Embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared with tenogenic induction in monolayer ESC cultures. The addition of TGF-β3 to the 3D cultures further up-regulates the expression of these genes and also induces the expression of mature tenocyte markers Tenomodulin and Thrombospondin-4. Our results show that when ESCs are exposed to the intrinsic forces exerted by a 3D culture environment, they express tendon-associated genes and proteins which are indicative of tenocyte lineage differentiation and that this effect is synergistically enhanced and accelerated by the addition of TGF-β3.
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transforming growth factor beta3 promotes tendon differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2013Co-Authors: Tom Barsby, Deborah J. GuestAbstract:Tendon injuries occur frequently in horses and have a poor capacity to regenerate, which leads to high re-injury rates. Equine Embryo-derived stem cells (ESCs) survive in high numbers in the injured horse tendon and we hypothesized that they differentiate into tenocytes in vivo. Immunocytochemistry revealed that in the injured horse tendon ESCs express the tendon progenitor marker scleraxis and that there is a local upregulation of the transforming growth factor-β (TGF-β) at the injury site. The aim of this study was to determine if TGF-β signaling was able to drive tenocyte differentiation by ESCs. Exposure of differentiating ESCs to TGF-β in vitro produced an upregulation of scleraxis at the gene and protein level with the greatest effect being produced in the presence of TGF-β3. TGF-β3 treatment of differentiating ESCs also promotes a significant upregulation of other tendon-associated genes and proteins suggesting it can promote ESC differentiation into tenocytes. Our results demonstrate that Equine E...
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transforming growth factor beta3 promotes tendon differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2013Co-Authors: Tom Barsby, Deborah J. GuestAbstract:Tendon injuries occur frequently in horses and have a poor capacity to regenerate, which leads to high re-injury rates. Equine Embryo-derived stem cells (ESCs) survive in high numbers in the injured horse tendon and we hypothesized that they differentiate into tenocytes in vivo. Immunocytochemistry revealed that in the injured horse tendon ESCs express the tendon progenitor marker scleraxis and that there is a local upregulation of the transforming growth factor-β (TGF-β) at the injury site. The aim of this study was to determine if TGF-β signaling was able to drive tenocyte differentiation by ESCs. Exposure of differentiating ESCs to TGF-β in vitro produced an upregulation of scleraxis at the gene and protein level with the greatest effect being produced in the presence of TGF-β3. TGF-β3 treatment of differentiating ESCs also promotes a significant upregulation of other tendon-associated genes and proteins suggesting it can promote ESC differentiation into tenocytes. Our results demonstrate that Equine ESCs can differentiate into a therapeutically relevant cell type and that TGF-β driven differentiation of ESCs may provide a model to study tendon development and better understand the transcriptional networks that are involved in Equine tendon cell differentiation from the early Embryonic stages.
K Hinrichs - One of the best experts on this subject based on the ideXlab platform.
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effect of warming method on Embryo quality in a simplified Equine Embryo vitrification system
Theriogenology, 2020Co-Authors: H S Canesin, I Ortiz, Alexandre Nascimento Rocha Filho, Renato Mayrink Salgado, J G Bromdeluna, K HinrichsAbstract:Abstract Equine Embryo vitrification is still not a well-established technique in Equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified Embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) Embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial Embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38 °C) for each of three warming systems: commercial warming solution (Kit); commercial Embryo holding medium (EHM) with decreasing concentrations of sucrose (EHM + SS); or EHM alone without added sucrose. Embryos (n = 9 to 14 per treatment) were cultured in vitro for 24 h, stained with DAPI, TUNEL, and fluorophore-labelled phalloidin, and evaluated for nucleus number, mitotic rate, apoptotic rate, and actin filament distribution. In Exp. 2, to survey Embryo viability in vivo, vitrified IVP blastocysts were shipped to an Embryo transfer facility, then warmed immediately before transfer to recipient mares, using the warming treatments associated with the nominally best (Kit-RT, Kit-38, EHM-RT) and poorest (EHM + SS-38) assessed Embryo quality in Exp. 1 (n = 7 to 8 per treatment). Subsequently, IVP blastocysts produced as part of our clinical program were vitrified and shipped, then warmed in Embryo holding medium at an Embryo transfer facility before transfer to recipient mares; fresh IVP Embryos were shipped and transferred as controls. In Exp. 1, Embryos increased significantly in diameter after culture (P 0.05) in the number of viable nuclei, apoptotic rate, or microfilament distribution among treatments, or between vitrified-warmed and Control Embryos. The mitotic rate was higher (P = 0.021) for Kit-RT (3.6%) when compared with the other treatment groups (1.5–2.0%). In Exp. 2, there was no difference (P > 0.05) in initial pregnancy (71.4–87.5%) or heartbeat (57.1%–85.7%) rates among warming treatments. In the clinical trial, there was no difference (P > 0.05) between vitrified-warmed and Control Embryos in initial pregnancy (90.9% and 66.6%, respectively) or heartbeat (81.8% and 66.6%, respectively) rates. These results indicate that a semi-closed vitrification system using commercially-available media, and incorporating warming in the field in a single step using commercial Embryo holding medium without cryoprotectants, can provide high pregnancy rates with IVP Equine Embryos.
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application of Embryo biopsy and sex determination via polymerase chain reaction in a commercial Equine Embryo transfer program in argentina
Reproduction Fertility and Development, 2019Co-Authors: Fernando L Riera, I Ortiz, Jaime E Roldan, J M Espinosa, J E Fernandez, K HinrichsAbstract:Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active Embryo transfer program. We evaluated results on biopsy of 459 Embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied Embryos (72% vs 73%) or for biopsied Embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped Embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller Embryos (70-85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive Embryos were confirmed as male and 78% of Y-DNA-negative Embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative Embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.
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morphokinetics of early Equine Embryo development in vitro using time lapse imaging and use in selecting blastocysts for transfer
Reproduction Fertility and Development, 2019Co-Authors: Niamh Lewis, K Hinrichs, Karen Schnauffer, Monica Morganti, Stephen Troup, C M ArgoAbstract:The use of time-lapse imaging (TLI) in the evaluation of morphokinetics associated with invitro developmental competence is well described for human, cattle and pig Embryos. It is generally accepted that Embryos that complete early cleavage sooner are more likely to form blastocysts and that timing of later events, such as blastocyst formation and expansion, are predictive of implantation potential and euploid status. In the horse, morphokinetics as a predictor of developmental competence has received little attention. In this study we evaluated the morphokinetics of early Equine Embryo development invitro for 144 oocytes after intracytoplasmic sperm injection and report the timings of blastocyst development associated with ongoing pregnancy for the first time. There was a tendency for time of cytoplasmic extrusion and first cleavage to occur earlier in the Embryos that went on to form blastocysts (n=19) compared with those that arrested, and for first cleavage to occur earlier in blastocysts that established pregnancies that were ongoing (n=4) compared with pregnancies that were lost (n=2). TLI was clinically useful in identifying blastocysts when evaluation of morphology on static imaging was equivocal.
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88 effect of bicarbonate co2 level during Embryo culture on Equine blastocyst rate after intracytoplasmic sperm injection
Reproduction Fertility and Development, 2017Co-Authors: Y H Choi, J G Bromdeluna, P Tinetti, K HinrichsAbstract:Equine Embryos appear to require a high glucose concentration for development to the blastocyst stage. The complete cell-culture medium, DMEM/F-12 (DM), which contains 17 mM glucose, has been widely used for Equine Embryo culture; however, in other species, high glucose during the early stages of Embryo development is detrimental. To avoid this, we initiated a 2-step system using a low-glucose human Embryo culture medium (Global) from Days 0 to 5 [Day 0 = day of intracytoplasmic sperm injection (ICSI)], with glucose (20 mM) added to the medium in the second step (Days 5 to 10; Choi et al. 2015 Reproduction 150, 31–41). We noted a high pregnancy loss rate (20%) in our clinical ICSI program (Hinrichs et al. 2014 J. Equine Vet. Sci. 34, 176), which used this 2-step Global system. Limited data are available on pregnancy with DM-produced Embryos, but in one study, the loss rate was 1/13 (7.7%; Choi et al. 2011 Reproduction 142, 529–538). It is possible that use of DM in the second step of culture would better support normal blastocyst development than does Global with added glucose. However, DM is typically used at 5% CO2, and Global at 6% CO2, so use of both media would necessitate 2 sets of incubators. In the present study, we explored the use of DM in the second step of a two-step Equine Embryo culture system, under different CO2 environments. Oocytes were collected from research mares via follicle aspiration and were held overnight before being matured in vitro for 30 h. All media included 10% fetal bovine serum. On Days 0 to 5 after ICSI, all Embryos were cultured in Global under 6% CO2 in mixed gas (5% O2 and remainder N2) at 38.2°C. In Experiment 1, on Day 5, Embryos were transferred to DM prepared according to our standard method, with 14.3 mM NaHCO3 and 5 mM NaOH, and were cultured in mixed gas at either 5% CO2 or 6% CO2. Five replicates were performed. In Experiment 2, DM was prepared by our standard method, or with 24.2 mM bicarbonate and no NaOH. When pH was measured using a pH meter after media were equilibrated overnight, this higher bicarbonate provided the same pH at 6% CO2 (pH 7.3), as was achieved with the standard DM preparation at 5% CO2. Six replicates were performed. In both experiments, blastocyst development was assessed on Days 7 to 10, and blastocyst rates were compared between treatments by Fisher’s exact test. In Experiment 1, blastocyst rates were 43%, 13/30 and 27%, 8/30 for the standard DM preparation in 5% and 6% CO2, respectively (P > 0.05). In Experiment 2, the blastocyst rates were 34%, 14/44 for the standard DM preparation at 5% CO2 and 43%, 19/44 for the high-bicarbonate DM at 6% CO2 (P > 0.05). We conclude that a 2-step Global-DM system can support Equine blastocyst production under a consistent CO2 environment (6%) if DM bicarbonate levels are adjusted to balance the increased CO2. This work was supported by the Clinical Equine ICSI Program, Texas A&M University, and by the Link Equine Research Endowment Fund, Texas A&M University.
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effect of medium variations zinc supplementation during oocyte maturation perifertilization ph and Embryo culture protein source on Equine Embryo development after intracytoplasmic sperm injection
Theriogenology, 2016Co-Authors: Y H Choi, H S Canesin, John R Gibbons, K HinrichsAbstract:Abstract Prospective studies were conducted to help define procedural factors affecting in vitro Embryo production via intracytoplasmic sperm injection (ICSI) of Equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in Embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% Equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P in vitro production of Equine Embryos.
Emma P Bavin - One of the best experts on this subject based on the ideXlab platform.
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three dimensional culture and transforming growth factor beta3 synergistically promote tenogenic differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2014Co-Authors: Tom Barsby, Emma P Bavin, Deborah J. GuestAbstract:The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of Equine Embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared...
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three dimensional culture and transforming growth factor beta3 synergistically promote tenogenic differentiation of Equine Embryo derived stem cells
Tissue Engineering Part A, 2014Co-Authors: Tom Barsby, Emma P Bavin, Deborah J. GuestAbstract:The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of Equine Embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared with tenogenic induction in monolayer ESC cultures. The addition of TGF-β3 to the 3D cultures further up-regulates the expression of these genes and also induces the expression of mature tenocyte markers Tenomodulin and Thrombospondin-4. Our results show that when ESCs are exposed to the intrinsic forces exerted by a 3D culture environment, they express tendon-associated genes and proteins which are indicative of tenocyte lineage differentiation and that this effect is synergistically enhanced and accelerated by the addition of TGF-β3.
Keith J. Betteridge - One of the best experts on this subject based on the ideXlab platform.
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5alpha reduced steroids are major metabolites in the early Equine Embryo proper and its membranes
Biology of Reproduction, 2015Co-Authors: James I. Raeside, Heather L. Christie, Keith J. BetteridgeAbstract:Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in Equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the Embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the Embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the Embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids--for example, 5alpha-dihydrotestosterone in male sexual differentiation--provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in early Embryonic development in horses and, more generally, in other species.
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Biosynthesis of oestrogen by the early Equine Embryo proper
Reproduction Fertility and Development, 2012Co-Authors: James I. Raeside, Heather L. Christie, R.o. Waelchli, Keith J. BetteridgeAbstract:The Embryo proper in early Equine pregnancy has recently been shown to have a remarkable capacity for metabolism of oestrogens. High concentrations of oestrogens in yolk-sac fluid could provide substrate for local metabolism in tissues of the Embryo proper and this activity could have significance for early development. Due to the high level of oestrogen metabolism in the Embryo proper we examined the possibility that it could also biosynthesise oestrogens. Conceptuses were collected in the fourth week of pregnancy (n = 23) and the Embryo was separated from extraEmbryonic tissues for incubation with [3H]androstenedione. Steroids were recovered from media by solid-phase extraction and eluted as unconjugated and conjugated fractions. Profiles of free and sulfoconjugated fractions, as well as the phenolic steroids extracted from them, were obtained by chromatography. Oestrone and oestradiol were seen clearly, indicating oestrogen biosynthesis, and the presence of more polar products, arising from metabolism of the primary oestrogens, gave further evidence that the Embryo was capable of oestrogen biosynthesis. Aromatase activity was also demonstrated by detection of tritium loss, as 3H2O, from incubations (n = 3) with [1β-3H]androstenedione. It is suggested that its oestrogen biosynthesis may have significance for the remarkable development of the vasculature in the Embryo proper at this stage.
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Estrogen metabolism by the Equine Embryo proper during the fourth week of pregnancy
REPRODUCTION, 2009Co-Authors: James I. Raeside, Heather L. Christie, R.o. Waelchli, Keith J. BetteridgeAbstract:Estrogen production by the trophoblast is considered important in early Equine pregnancy and leads to high concentrations in yolk-sac (Y-S) fluid. The Embryo proper is a potential site for their action. We examined estrogen metabolism in the Embryo proper because some actions of estrogens are derived from locally formed metabolites. The Embryo proper, as well as separated extraEmbryonic tissues, of conceptuses collected about day 25 of pregnancy, were incubated with (3)[H]-estrone (E(1)) and (3)[H]-estradiol (E(2)). Steroids were recovered from media by solid-phase extraction and eluted separately as unconjugated and conjugated fractions. Profiles of free and sulfo-conjugated fractions were obtained by HPLC. Some differences and similarities were noted for the Embryo proper as compared to the extraEmbryonic tissues. No reduction of E(1) to E(2) was noted for the Embryo proper and allantois, but some was seen with the bilaminar Y-S wall. Less conversion of E(2) to E(1) occurred in the Embryo proper than in the extraEmbryonic tissues. Profiles for hydrolyzed sulfates from incubation of the Embryo proper were very similar for both substrates, mainly with E(1) present. Thus, low levels of reductase and high levels of oxido- activities were apparent for the 17beta-hydroxysteroid dehydrogenase enzymes. Further evidence of an active role for the Embryo proper was seen as minor, polar products, and an unknown compound eluting between E(2) and E(1). These findings show, for the first time, that the Embryo proper can metabolize estrogens that are found in Y-S fluid - a function of potential significance at this stage in its development.