Introduction and aim. The definition of enriched culture conditions for oocytes that have lost their supporting cumulus cells might be helpful for limiting the discarding of fresh cumulus-denuded oocytes (CDOs) from in vitro embryo production (IVEP), and for increasing the performances of thawed ones, generally deprived of surrounding cumulus cells. In a previous study, the encapsulation of CDOs in a three-dimensional system with cumulus-oocyte complexes (COCs) as companion cells, resulted in the increase of their viability and in the slight improvement of their meiotic competence in vitro (1). To evaluate whether the beneficial effect of companion COCs and 3D conditions during in vitro maturation could improve the subsequent embryo development, feline CDOs were matured with companion COCs in barium alginate capsules (3D system) or in traditional drops (2D system) followed by fertilization and embryo development in 3D or 2D conditions. Materials and methods. Grade I COCs (n=330; 8 replicates) were collected from ovaries of domestic queens; 115 COCs were mechanically deprived of cumulus cells with a small-bore pipette. Cumulus-denuded oocytes (CDOs) with companion COCs, or COCs alone as control group were encapsulated in barium alginate (Sigma Chemical Co., MO, USA) capsules (3D system) or placed in 100 l drops (2D system) and in vitro matured in Quinn’s Advantage Protein Plus Blastocyst medium (SBP, Sage®, with 5% FCS; 10 µg/mL FSH, 10 µg/mL LH; NIH) for 24 h in a controlled atmosphere (38.5°C and 5% CO2 in air). Fresh epididymal spermatozoa were selected by swim-up processing in SBP medium and the oocytes were inseminated with 0.75-1 x 106 motile spermatozoa/ml. At 18-24 h after in vitro fertilization, presumptive zygotes from CDOs, COCs companion and COCs control were in vitro cultured for 7 days in SBP medium in 3D or 2D system according to in vitro maturation conditions. Embryo stages were determined after fixation and DAPI (4′,6-diamidino-2-phenylindole; Vectashield®) staining. Data were analyzed by Chi-square test (p<0.05). Results. The results showed that the overall proportion of embryos (total n. embryos/total n. oocytes) was similar (p>0.05) when 3D and 2D culture systems were used during oocyte maturation and embryo culture (CDOs: 12.5% vs. 16.9%; COCs companion: 52.6% vs. 49.1%; COCs control: 31.1% vs. 21.1%). However, the embryo development of COCs matured as companion cells of CDOs was significantly higher than that of CDOs themselves and of COCs cultured alone (control) in both 3D (52.6% vs. 12.5%, p<0.00005, and vs. 31.1%, p<0.05, respectively) and 2D system (49.1% vs. 16.9%, p<0.0005, and vs. 21.1%, p<0.005, respectively). No significant differences were observed in the percentages of late embryo stages (total n. morulae plus blastocysts/total n. oocytes) in 3D and 2D culture (CDOs: 7.1% vs. 10.2%; COCs companion: 40.4% vs. 38.6%; COCs control: 6.7% vs. 19.3%), whereas COCs companion showed higher results compared to CDOs themselves and COCs cultured alone in both 3D (40.4% vs. 7.1%, p<0.00001, and vs. 6.7%, p<0.00005, respectively) and 2D system (38.6% vs. 10.2%, p<0.0001, and vs. 19.3%, p<0.05, respectively). Conclusions. The 3D maturation system with companion COCs in SBP medium did not give the expected results in supporting the embryo development of feline CDOs. However, COCs companion benefit from the co-culture with CDOs both in 3D and 2D maturation, presumably thanks to the oocyte secrete factors (OSFs) released by the CDOs, that support the specialized cumulus cells microenvironment necessary for the acquisition of full developmental competence, as already reported in other species (2-3-4). References. 1) Morselli et al. Proc. 17th Ann Congr EVSSAR, Wroclaw (Poland), 2014, p. 198. 2) Luciano et al. Mol Reprod Dev 2005;71:389-97. 3) Hussein et al. Dev Biol 2006;296:514-21 4) Gilchrist et al. Hum Reprod Update 2008;14:159-77.
Embryo development of cumulus-denuded feline oocytes in vitro matured in a three-dimensional alginate scaffold / M.G. Morselli, G.C. Luvoni, P. Comizzoli. ((Intervento presentato al 18. convegno EVSSAR tenutosi a Hannover nel 2015.
Embryo development of cumulus-denuded feline oocytes in vitro matured in a three-dimensional alginate scaffold
M.G. Morselli
;G.C. LuvoniPenultimo
;
2015
Abstract
Introduction and aim. The definition of enriched culture conditions for oocytes that have lost their supporting cumulus cells might be helpful for limiting the discarding of fresh cumulus-denuded oocytes (CDOs) from in vitro embryo production (IVEP), and for increasing the performances of thawed ones, generally deprived of surrounding cumulus cells. In a previous study, the encapsulation of CDOs in a three-dimensional system with cumulus-oocyte complexes (COCs) as companion cells, resulted in the increase of their viability and in the slight improvement of their meiotic competence in vitro (1). To evaluate whether the beneficial effect of companion COCs and 3D conditions during in vitro maturation could improve the subsequent embryo development, feline CDOs were matured with companion COCs in barium alginate capsules (3D system) or in traditional drops (2D system) followed by fertilization and embryo development in 3D or 2D conditions. Materials and methods. Grade I COCs (n=330; 8 replicates) were collected from ovaries of domestic queens; 115 COCs were mechanically deprived of cumulus cells with a small-bore pipette. Cumulus-denuded oocytes (CDOs) with companion COCs, or COCs alone as control group were encapsulated in barium alginate (Sigma Chemical Co., MO, USA) capsules (3D system) or placed in 100 l drops (2D system) and in vitro matured in Quinn’s Advantage Protein Plus Blastocyst medium (SBP, Sage®, with 5% FCS; 10 µg/mL FSH, 10 µg/mL LH; NIH) for 24 h in a controlled atmosphere (38.5°C and 5% CO2 in air). Fresh epididymal spermatozoa were selected by swim-up processing in SBP medium and the oocytes were inseminated with 0.75-1 x 106 motile spermatozoa/ml. At 18-24 h after in vitro fertilization, presumptive zygotes from CDOs, COCs companion and COCs control were in vitro cultured for 7 days in SBP medium in 3D or 2D system according to in vitro maturation conditions. Embryo stages were determined after fixation and DAPI (4′,6-diamidino-2-phenylindole; Vectashield®) staining. Data were analyzed by Chi-square test (p<0.05). Results. The results showed that the overall proportion of embryos (total n. embryos/total n. oocytes) was similar (p>0.05) when 3D and 2D culture systems were used during oocyte maturation and embryo culture (CDOs: 12.5% vs. 16.9%; COCs companion: 52.6% vs. 49.1%; COCs control: 31.1% vs. 21.1%). However, the embryo development of COCs matured as companion cells of CDOs was significantly higher than that of CDOs themselves and of COCs cultured alone (control) in both 3D (52.6% vs. 12.5%, p<0.00005, and vs. 31.1%, p<0.05, respectively) and 2D system (49.1% vs. 16.9%, p<0.0005, and vs. 21.1%, p<0.005, respectively). No significant differences were observed in the percentages of late embryo stages (total n. morulae plus blastocysts/total n. oocytes) in 3D and 2D culture (CDOs: 7.1% vs. 10.2%; COCs companion: 40.4% vs. 38.6%; COCs control: 6.7% vs. 19.3%), whereas COCs companion showed higher results compared to CDOs themselves and COCs cultured alone in both 3D (40.4% vs. 7.1%, p<0.00001, and vs. 6.7%, p<0.00005, respectively) and 2D system (38.6% vs. 10.2%, p<0.0001, and vs. 19.3%, p<0.05, respectively). Conclusions. The 3D maturation system with companion COCs in SBP medium did not give the expected results in supporting the embryo development of feline CDOs. However, COCs companion benefit from the co-culture with CDOs both in 3D and 2D maturation, presumably thanks to the oocyte secrete factors (OSFs) released by the CDOs, that support the specialized cumulus cells microenvironment necessary for the acquisition of full developmental competence, as already reported in other species (2-3-4). References. 1) Morselli et al. Proc. 17th Ann Congr EVSSAR, Wroclaw (Poland), 2014, p. 198. 2) Luciano et al. Mol Reprod Dev 2005;71:389-97. 3) Hussein et al. Dev Biol 2006;296:514-21 4) Gilchrist et al. Hum Reprod Update 2008;14:159-77.Pubblicazioni consigliate
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