Effect of a three-dimensional co-culture system on feline oocyte competence to develop into an embryo

Three-dimensional (3D) in vitro culture has been developed to improve the full competence of mammalian oocytes. These culture systems mimic the in vivo spatial arrangement and physiological conditions for the oocytes. In addition, the co-culture with companion cells, such as cumulus-denuded oocytes (CDOs) or cumulus cells (CCs) clumps, has been shown to improve the oocyte maturation and subsequent embryo development in different species, including domestic cat [1,2,3]. The aim of this study was to investigate the effect of an enriched culture system (3D barium alginate microcapsules associated with companion CDOs) on the in vitro embryo development of feline cumulus-oocyte complexes (COCs). Grade I COCs (n=545; 8 to 12 replicates) were collected from adult ovaries and 187 were mechanically deprived of CCs to obtain the CDOs. The COCs were co-cultured with CDOs in barium alginate (Sigma Chemical Co., MO, USA) microcapsules (3D system) in Quinn\u2019s Advantage Protein Plus Blastocyst medium (SBP, SAGE\uae In Vitro Fertilization, Trumbull, Connecticut, USA) with 75 UI FSH + 75 UI LH (Menogon\uae, Ferring Pharmaceuticals, Switzerland), 10 ng/ml of epidermal growth factor (EGF), antibiotics (AB) and 0.6 mM cysteine, in a controlled atmosphere (38.5\ub0C and 5% CO2 in air) for 24 h. Control groups of COCs alone were cultured separately. For in vitro fertilization, chilled epididymal spermatozoa were selected by swim-up treatment in SBP and the oocytes were inseminated with 0.75-1 x 106 motile spermatozoa/ml. At 18-24 h post-insemination, presumptive zygotes were in vitro cultured for 7 days in 3D microcapsules in SBP with 5% of FCS and AB (National Institutes of Health, Bethesda, MD, USA). The oocyte meiotic progression after IVM and embryo stages at the end of culture were determined by fixation and staining with bis-benzimide (Hoechst 33342; Sigma). Data were analyzed by Chi-square test (p<0.05). The meiosis resumption of COCs (in co-culture: 86.1% and cultured alone: 88.5%) was significantly higher than that of CDOs (40.2%; p<0.00001). The COCs cultured separately showed the highest full maturational (TI-MII) rates (p<0.00001) compared to those of COCs co-cultured with CDOs (74.2% vs 37.5%) and of CDOs themselves (74.2% vs 12.5%). Inversely, the proportions of late embryos stages (total n. morulae and blastocysts/total n. cleaved embryos) of COCs co-cultured with CDOs were higher than those of COCs cultured separately (77.6% vs 53.8%), and no difference was found between CDOs and COCs control (58.8% vs 53.8%; p=0.05). The 3D barium alginate microcapsules in association with companion CDOs were a suitable enriched culture system to improve the developmental competence of domestic cat COCs. The presence of denuded oocytes and related secreted factors supported the achievement of COCs cytoplasmic maturation, essential for further embryo development. Moreover, the CDOs themselves, known as low competence gametes, also benefited from this condition, as their late embryo stages were similar to those of COCs control. [1] Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum Reprod Update 2008;14:159-177. [2] Chigioni S, Perego L, Luvoni GC. Companion cumulus-oocyte complexes for in vitro culture of denuded oocytes in the cat. Proc. 4th Ann Congr EVSSAR, Amsterdam (The Netherlands), 2005, pp. 24-25. [3] Godard N, Pukazhenthi BS, Wildt DE, et al. Paracrine factors from cumulus-enclosed oocytes ensure the successful maturation and fertilization in vitro of denuded oocytes in the cat model. Fertil Steril 2009;91:2051-2060

Long-term storage of gametes and gonadal tissues at room temperatures: the end of the ice age?

Long-term preservation of viable spermatozoa, eggs, embryos, and gonadal tissues of good quality is essential in human reproductive medicine and for the population management of livestock, laboratory, and wild species. Instead of using freezing temperatures, encouraging findings indicate that structures and functions of gametes or gonadal tissues can be suspended in trehalose glass after dehydration and then preserved at supra-zero temperatures. As a new era in fertility preservation and biobanking is about to start, the advantages, needs, and implications of germplasm storage at room temperatures must be carefully examined. Although very promising, the development of alternate biobanking strategies does not necessarily mean that the end of the “ice age” (cryopreservation) is near

Embryo development of cumulus-denuded feline oocytes in vitro matured in a three-dimensional alginate scaffold

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 \uf06dl drops (2D system) and in vitro matured in Quinn\u2019s Advantage Protein Plus Blastocyst medium (SBP, Sage\uae, with 5% FCS; 10 \ub5g/mL FSH, 10 \ub5g/mL LH; NIH) for 24 h in a controlled atmosphere (38.5\ub0C 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\u2032,6-diamidino-2-phenylindole; Vectashield\uae) 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

The nuclear and developmental competence of cumulus-oocyte complexes is enhanced by three-dimensional coculture with conspecific denuded oocytes during in vitro maturation in the domestic cat model

The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24\ua0hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7\ua0days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required

Assessment of in vitro fertility of deer spermatozoa by heterologous ivf with zona-free bovine oocytes

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