- Email: [email protected]
Maturation Condition alters the Cumulus Proteome of Equine Oocytes
Abstracts / Journal of Equine Veterinary Science 41 (2016) 51e84
into 3 groups according to the chromatin conformation: (1) MI (meiotic progression including Metaphase I and Anaphase I), (2) MII (matured including Telophase I and Metaphase II) and (3) oocytes including germinal vesicle stage and degenerate oocytes. A generalized linear model was built in SAS version 9.2, using PROC-GLM to determine the differences in maturation rate (MII %), meiotic progression (MI%) and degeneration between groups. Data are reported as model least square means with standard errors (LSM ± SE). Significance and tendency were declared at P< 0.05 and 0.05 < P < 0.1 respectively. In six replicates, 905 oocytes were randomly assigned to four groups: oocytes placed directly in maturation condition (N¼159) and oocytes stored at 4 C (N¼259), 17 C (N¼249) or 22-25 C (N¼238) overnight. After 24h of IVM, maturation rate was similar for all groups (63%, 63%, 68% and 60% at 4 C, 17 C and 25 C and direct maturation, respectively, P>0.05), suggesting that COCs can be stored in conventional 4 C transport conditions. In our preliminary studies, COCs were held at 4 C followed by 24h of IVM and mature oocytes were fertilized using ICSI. Twenty-three injected developed into 4 confirmed blastocysts.
Acknowledgments Funded by an UGent IWETO grant. With thanks to Isabel Lemahieu and Petra Van Damme for technical assistance.
62 Maturation Condition alters the Cumulus Proteome of Equine Oocytes F. Huwiler 1, C. Fortes 2, J. Grossmann 2, B. Roschitzki 2, U. Bleul 1, J. Walter 1, * 1 Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland; 2 Functional Genomics Center Zurich, University and ETH Zurich, 8057 Zurich, Switzerland *Corresponding author: [email protected] Maturation is the first decisive step for the production of embryos in-vitro. The developmental competence of equine oocytes matured in-vitro is tremendously impaired compared to their invivo matured counterparts (Hinrichs et. al; Mol Reprod Dev 2010;77:651-661). Successful conventional in-vitro fertilization (IVF) in the horse was only successful using in-vivo matured oocytes (Palmer et al; J Reprod and Fertil Suppl 1991;44:375e384). This illustrates the need for improved maturation media that mimic the physiological conditions, which may contribute to overcome the failure of IVF in the horse. Through the close metabolic communication between cumulus cells and oocytes via tight and gap junctions, cumulus cells represent a unique source to study metabolism of equine cumulus oocyte complexes (COCs). The aim of this study was to investigate alterations in the proteome of cumulus complexes (CC) corresponding to single Metaphase II oocytes matured in-vivo and CC matured in-vitro (n¼7 in each group). COCs matured in-vivo were collected from mares in oestrus (follicle >35mm, uterine oedema). These mares were slaughtered 30 hours after injection of 2500 I.U. of human chorionic gonadotropin (hCG). COCs matured in-vitro were collected from regular slaughtered mares in dioestrus. They were incubated in DMEM based maturation medium for 30h. CC were separated from their oocyte, washed in PBS and stored in liquid nitrogen. The cumulus free oocyte was examined for extrusion of the first polar body, to assure successful maturation. Only successful matured oocytes were examined for this study. Proteome analysis was conducted by a mass spectrometry (MS) based approach. An adapted filter-aided sample
79
preparation protocol was used for cell lysis and protein digestion (Wisniewski et. al; Nat Methods 2009;6:359e62). MS was performed by nano-HPLC MS/MS and Progenesis QI for Proteomics software (Nonlinear Dynamics) was used for data analysis. 2060 quantifiable proteins were detected in the 14 samples, of which 242 proteins were differently expressed between the groups (fold change > 2, p 0.05). 102 proteins were upregulated in the in vivo matured MII COCs and 140 in the in vitro matured. Enrichment analysis using the String-Database (http://string-db.org) revealed a significantly overrepresented complement and coagulation cascade pathway in cumulus of in-vivo matured COCs. Part of this pathway is made up of several proteins of the innate immune system, which plays an important role in the ovulatory process. Proteins involved in fertilization belonged to the ones overexpressed in in-vivo matured COCs: cysteine rich secretory protein (CRISPLD2; F6SS57_HORSE) and Zona pellucida sperm binding protein 3 (ZP3R, F6ZD61_HORSE); as well as important constituents of the extracellular matrix: versican core protein (VCAN; F7DY34_HORSE) and inter-alpha-trypsin inhibitor heavy chain (ITIH1-4, F6QX36_HORSE, F7CTJ3_HORSE, F7AE68_HORSE, F6R3F4_HORSE). Several proteins overexpressed in the in-vitro matured group are connected to heat shock or oxidative stress. This study characterizes major alterations in the cumulus proteome through the ex-vivo maturation process of COCs. These findings may contribute to improvements in the limited success of equine IVF. Further investigations on the dynamics of the differentially expressed proteins during the maturation procedure are necessary to corroborate these results and transfer them to the clinical IVF lab.
63 Toxicity of a vitrification protocol for equine immature oocytes Heloísa S. Canesin*, Joao G. Brom-de-Luna, Young-Ho Choi, Katrin Hinrichs College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, US, 77843-4466 *Corresponding author: [email protected] Equine oocytes are difficult to obtain for many research laboratories. Cryopreservation of immature oocytes is generally less successful than is that of mature oocytes, but would allow collection of oocytes at a location in which no culture facilities were available, for transportation to a research laboratory. We evaluated methods for vitrification of immature equine oocytes. Oocytes were recovered from immature follicles of live mares and held overnight at room temperature should ask authors to specify conditions. In Experiment 1, oocytes were randomly allotted to either control (CON) or vitrification (VIT) groups. CON oocytes were cultured for in vitro maturation (IVM), then mature oocytes were subjected to ICSI and embryo culture. VIT oocytes were placed in base solution consisting of 80% M199-Hanks' salts and 20% FBS (MH), then equilibrated in three steps until reaching 7.5% DMSO and 7.5% ethylene glycol in a total time of 14 min. Oocytes were then placed in vitrification solution (MH with 15% DMSO, 15% ethylene glycol, and 0.5 M sucrose) for 1 min, then 5 to 10 oocytes were transferred to a 75-mm sterile stainless steel mesh. The mesh was blotted to absorb excess medium, then plunged in liquid nitrogen. The oocytes were warmed at 37oC in MH solution with 1.25 M sucrose for 1 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and then placed into MH. Oocytes were then subjected to IVM, ICSI and embryo culture as for CON. Differences in maturation, cleavage and blastocyst rates between groups were analyzed by Fisher's exact test.
into 3 groups according to the chromatin conformation: (1) MI (meiotic progression including Metaphase I and Anaphase I), (2) MII (matured including Telophase I and Metaphase II) and (3) oocytes including germinal vesicle stage and degenerate oocytes. A generalized linear model was built in SAS version 9.2, using PROC-GLM to determine the differences in maturation rate (MII %), meiotic progression (MI%) and degeneration between groups. Data are reported as model least square means with standard errors (LSM ± SE). Significance and tendency were declared at P< 0.05 and 0.05 < P < 0.1 respectively. In six replicates, 905 oocytes were randomly assigned to four groups: oocytes placed directly in maturation condition (N¼159) and oocytes stored at 4 C (N¼259), 17 C (N¼249) or 22-25 C (N¼238) overnight. After 24h of IVM, maturation rate was similar for all groups (63%, 63%, 68% and 60% at 4 C, 17 C and 25 C and direct maturation, respectively, P>0.05), suggesting that COCs can be stored in conventional 4 C transport conditions. In our preliminary studies, COCs were held at 4 C followed by 24h of IVM and mature oocytes were fertilized using ICSI. Twenty-three injected developed into 4 confirmed blastocysts.
Acknowledgments Funded by an UGent IWETO grant. With thanks to Isabel Lemahieu and Petra Van Damme for technical assistance.
62 Maturation Condition alters the Cumulus Proteome of Equine Oocytes F. Huwiler 1, C. Fortes 2, J. Grossmann 2, B. Roschitzki 2, U. Bleul 1, J. Walter 1, * 1 Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland; 2 Functional Genomics Center Zurich, University and ETH Zurich, 8057 Zurich, Switzerland *Corresponding author: [email protected] Maturation is the first decisive step for the production of embryos in-vitro. The developmental competence of equine oocytes matured in-vitro is tremendously impaired compared to their invivo matured counterparts (Hinrichs et. al; Mol Reprod Dev 2010;77:651-661). Successful conventional in-vitro fertilization (IVF) in the horse was only successful using in-vivo matured oocytes (Palmer et al; J Reprod and Fertil Suppl 1991;44:375e384). This illustrates the need for improved maturation media that mimic the physiological conditions, which may contribute to overcome the failure of IVF in the horse. Through the close metabolic communication between cumulus cells and oocytes via tight and gap junctions, cumulus cells represent a unique source to study metabolism of equine cumulus oocyte complexes (COCs). The aim of this study was to investigate alterations in the proteome of cumulus complexes (CC) corresponding to single Metaphase II oocytes matured in-vivo and CC matured in-vitro (n¼7 in each group). COCs matured in-vivo were collected from mares in oestrus (follicle >35mm, uterine oedema). These mares were slaughtered 30 hours after injection of 2500 I.U. of human chorionic gonadotropin (hCG). COCs matured in-vitro were collected from regular slaughtered mares in dioestrus. They were incubated in DMEM based maturation medium for 30h. CC were separated from their oocyte, washed in PBS and stored in liquid nitrogen. The cumulus free oocyte was examined for extrusion of the first polar body, to assure successful maturation. Only successful matured oocytes were examined for this study. Proteome analysis was conducted by a mass spectrometry (MS) based approach. An adapted filter-aided sample
79
preparation protocol was used for cell lysis and protein digestion (Wisniewski et. al; Nat Methods 2009;6:359e62). MS was performed by nano-HPLC MS/MS and Progenesis QI for Proteomics software (Nonlinear Dynamics) was used for data analysis. 2060 quantifiable proteins were detected in the 14 samples, of which 242 proteins were differently expressed between the groups (fold change > 2, p 0.05). 102 proteins were upregulated in the in vivo matured MII COCs and 140 in the in vitro matured. Enrichment analysis using the String-Database (http://string-db.org) revealed a significantly overrepresented complement and coagulation cascade pathway in cumulus of in-vivo matured COCs. Part of this pathway is made up of several proteins of the innate immune system, which plays an important role in the ovulatory process. Proteins involved in fertilization belonged to the ones overexpressed in in-vivo matured COCs: cysteine rich secretory protein (CRISPLD2; F6SS57_HORSE) and Zona pellucida sperm binding protein 3 (ZP3R, F6ZD61_HORSE); as well as important constituents of the extracellular matrix: versican core protein (VCAN; F7DY34_HORSE) and inter-alpha-trypsin inhibitor heavy chain (ITIH1-4, F6QX36_HORSE, F7CTJ3_HORSE, F7AE68_HORSE, F6R3F4_HORSE). Several proteins overexpressed in the in-vitro matured group are connected to heat shock or oxidative stress. This study characterizes major alterations in the cumulus proteome through the ex-vivo maturation process of COCs. These findings may contribute to improvements in the limited success of equine IVF. Further investigations on the dynamics of the differentially expressed proteins during the maturation procedure are necessary to corroborate these results and transfer them to the clinical IVF lab.
63 Toxicity of a vitrification protocol for equine immature oocytes Heloísa S. Canesin*, Joao G. Brom-de-Luna, Young-Ho Choi, Katrin Hinrichs College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, US, 77843-4466 *Corresponding author: [email protected] Equine oocytes are difficult to obtain for many research laboratories. Cryopreservation of immature oocytes is generally less successful than is that of mature oocytes, but would allow collection of oocytes at a location in which no culture facilities were available, for transportation to a research laboratory. We evaluated methods for vitrification of immature equine oocytes. Oocytes were recovered from immature follicles of live mares and held overnight at room temperature should ask authors to specify conditions. In Experiment 1, oocytes were randomly allotted to either control (CON) or vitrification (VIT) groups. CON oocytes were cultured for in vitro maturation (IVM), then mature oocytes were subjected to ICSI and embryo culture. VIT oocytes were placed in base solution consisting of 80% M199-Hanks' salts and 20% FBS (MH), then equilibrated in three steps until reaching 7.5% DMSO and 7.5% ethylene glycol in a total time of 14 min. Oocytes were then placed in vitrification solution (MH with 15% DMSO, 15% ethylene glycol, and 0.5 M sucrose) for 1 min, then 5 to 10 oocytes were transferred to a 75-mm sterile stainless steel mesh. The mesh was blotted to absorb excess medium, then plunged in liquid nitrogen. The oocytes were warmed at 37oC in MH solution with 1.25 M sucrose for 1 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and then placed into MH. Oocytes were then subjected to IVM, ICSI and embryo culture as for CON. Differences in maturation, cleavage and blastocyst rates between groups were analyzed by Fisher's exact test.