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Oocytes vitribanking in the domestic cat model: developmental competence in 3D culture
No full textIn the domestic cat model, vitrified oocytes (VOs) produced promising results, however their potential to develop into late stage embryos is often impaired.
Instead of culturing VOs in two-dimensional (2D) microdrops of medium (conventional system), the use of three-dimensional (3D) scaffolds could better maintain their functional integrity, contributing to oocyte in vitro outcomes. Therefore, the aim of this study was to investigate survival and embryo development (oocyte competence) of feline VOs cultured in 3D barium alginate microcapsules.
Morphologically selected immature cumulus-oocyte complexes (COCs, n=250) were vitrified by Cryotop method, and viable oocytes after warming were in vitro matured for 24 hours in 2D (2D VOs) or 3D system, with fresh companion COCs in 1:1 ratio [3D VOs(+)] or without [3D VOs(-)]. After in vitro fertilization, presumptive zygotes were cultured for 7 days in 3D or 2D system, according to the maturation conditions. Viability of VOs after warming and number of blastomeres of embryos were assessed by fluorescent stainings (fluorescein diacetate/propidium iodide and bis-benzimide, respectively). Arcsin transformed data were analysed by one-way ANOVA followed by Tukey test, and the level of significance was set at p<0.05.
After warming, 91.2% VOs were viable. Embryos were successfully obtained at similar proportions in each group [mean ± SD: 3D VOs(+) 0.18 ± 0.17; 3D VOs(-) 0.17 ± 0.12; 2D VOs 0.15 ± 0.09; p>0.05], but the use of the 3D system together with companion COCs was determinant for their development, since 3D VOs(+) were the only group to reach the blastocyst stage (mean ± SD: 0.01 ± 0.03, day 7). Degeneration rates did not differ among groups.
These positive results prompt the use of 3D enriched culture systems for VOs to exploit this valuable germplasm and widen the potential of vitribanking
Granulosa cells & three-dimensional scaffold: towards a follicle-like structure in the domestic cat
No full textThe in vitro development of feline oocytes is still suboptimal, especially in case of low competence gametes, such as cryopreserved or denuded oocytes, valuable resources for fertility preservation purposes. Modifications to media composition alone have not led to truly satisfactory results; therefore, since physical factors can positively influence the chemical conditions (1), a three-dimensional (3D) follicle-like structure could enhance meiosis resumption and further embryo development in vitro of poorly competent oocytes. In the domestic cat, the establishment of granulosa cells (GCs) culture was performed in a bidimensional (2D) system, and steroidogenesis was revealed along culture (2). However, since the follicular environment is three-dimensionally organized, the encapsulation of GCs in a 3D matrix might improve their self-organization, proliferation and endocrine functions. With the final aim of using these follicle-like structures for low competence oocyte IVM, functionality and efficiency of GCs encapsulated in biocompatible 3D matrix were evaluated during culture, and estradiol (E2) and progesterone (P4) concentrations were compared to those obtained from GCs cultured in a 2D monolayer. Cat granulosa cells were collected from isolated ovaries by enzymatic denudation (hyaluronidase 80 IU) of fresh COCs. Cells were processed as in (2) and then re-suspended in culture medium [CM=TCM199 supplemented with 0.1% BSA, 5% FBS, 0.5 IU FSH + 0.5 IU LH, 10 μg/mL 22(R)-hydroxycholesterol, 25 ng/ml epidermal growth factor, 0.1% ITS premix (1.0 μg/mL insulin; 0.55 μg/mL transferrin; 0.5 ng/mL sodium selenite), 10-7 M androstenedione]. Granulosa cells were in vitro cultured in a controlled atmosphere for 6 days in 3D follicle-like structures [obtained by one-step encapsulation in barium alginate (3)], or in 2D system (multiwell plates). Aliquots of conditioned CM were collected on days 2 and 6 and stored at -20°C until hormonal (E2 and P4) determination with an Enzyme Linked Fluorescent Assay (ELFA, MiniVIDAS®, Biomerieux, France). Log transformed hormonal concentrations were analyzed by a two-way ANOVA followed by Tukey test; the level of significance was set at p<0.05. Differences in hormonal production among days and systems of GCs culture were observed. Estradiol and P4 concentrations differed significantly during time, resulting higher on day 6 compared to day 2 (p=0.018) and day 0 (p<0.001) of culture. Steroidogenesis was obtained in 3D follicle-like structures as well as in 2D monolayers, although not to the same extent; the systems differed significantly only on day 6 (P4: 2.44 ± 1.28 ng/ml in 3D and 610 ± 607 ng/ml in 2D, p<0.001; E2: 173 ± 145 pg/ml in 3D and 26,709 ± 17,921 pg/ml in 2D, p=0.02). Present results underline that GCs in 3D microcapsules maintained their physiological features, as hormonal secretion. To test whether these 3D conditions could act as proper follicle-like structures, further experiments will be focused on the assessment of their nursing ability for the in vitro maturation of low competence oocytes.
This research project was funded by EVSSAR Grant 2017.
References
(1) Luvoni GC. The never ending search of an ideal culture system for domestic cat oocytes and embryos. Reprod Domest Anim 2018; accepted.(2) Simsek O, Arikan S. Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro. Anim Reprod 2015;12:931-938.
(3) Vigo D, Villani S, Faustini M, et al. Follicle-like model by granulosa cell encapsulation in a barium alginateprotamine membrane. Tissue Eng 2005;11:709-14
Overcoming flat biology: three-dimensional follicle-like structures for domestic cat vitrified oocytes
No full textFollicle-like structures are a combination of living cells and semipermeable 3D matrices, in which physical (3D systems) and chemical (cell-secreted molecules) factors create an in vivolike microenvironment for the in vitro development of female gametes (1, 2). This artificial and viable milieu, successfully applied for fresh oocytes, could be even more useful for stressed gametes, as the low competence vitrified oocytes (VOs). Key players of fertility preservation efforts, VOs poorly achieve full maturation because their inner competence is severely impaired by unavoidable cryodamages also involving their surrounding cumulus cells. The replacement of damaged somatic compartment with species-specific follicle-like structures could restore the developmental competence of VOs. Thus, in this work the creation of a follicle-like structure of granulosa cells (GCs) in 3D barium alginate microcapsules was aimed at improving the nuclear competence of domestic cat VOs. Therefore, a comparison with “flat” GCs culture (monolayer) was performed to test whether 3D spatial organization could be more adequate in preserving GCs nursing ability for stressed gametes. Isolated ovaries (n. 22) from domestic queens were minced to retrieve fresh oocytes that were enzymatically denuded (hyaluronidase 80IU) to collect GCs. After purification (3), GCs were suspended in culture medium [CM=TCM199 supplemented with 0.1% BSA, 5% FBS, 0.5 IU FSH + 0.5 IU LH, 10 μg/mL 22(R)-hydroxycholesterol, 25 ng/ml epidermal growth factor, 0.1% ITS premix (1.0 μg/mL insulin; 0.55 μg/mL transferrin; 0.5 ng/mL sodium selenite), 10- 7 M androstenedione], and the one-step encapsulation technique in barium alginate (2) was performed to obtain the 3D follicle-like structures. Granulosa cells capsules (3D system) or monolayers (2D) were in vitro cultured in CM at controlled atmosphere (38.5°C and 5% CO2 in air) for 2 or 6 days and used as artificial milieu for VOs IVM. Vitrified oocytes were obtained by Cryotop protocol (Kitazato Co., Fujinomiya, Japan) from 127 fresh COCs, and after warming only viable gametes (stained by FDA/PI, n. 112) were in vitro matured (24 h) in 3D and 2D system, either on day 2 or on day 6 of GCs culture. Chromatin configurations were determined by bis-benzimide (Hoechst 33342; Sigma) staining, and data were analyzed by Chi-square test, with the level of significance set at p≤0.05. Very encouraging meiosis resumption rates were obtained in VOs matured in 3D follicle-like structures (45.5%) and monolayer (56.7%) on day 2 of GCs culture. Prolonged culture of GCs for 6 days severely impaired the nursing ability of monolayer (21.7%, p=0.007), but not that of 3D follicle-like structures (26.7%, p=0.07), that supported higher proportions of fullmaturational (TI-MII) stages (17.4%) than GCs cultured for 2 days (0%). Culturing GCs for 2 days allowed lower VOs degeneration rates than 6 days (p=0.001). The ideal culture environment for low competence VOs has to combine physical and chemical factors and 3D follicle-like structures gave promising results for the restoration of VOs fertility potential, but further improvements are needed to promote GCs polarization and self-assembly into tissue-like structures.
This research project was funded by EVSSAR Grant 2017.
(1) Luyckx V, Dolmans MM, Vanacker J et al. A new step towards the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold. Fertil Steril, 2014; 101: 1149–56.
(2) Vigo D, Villani S, Faustini M et al. Follicle-like model by granulosa cell encapsulation in a barium alginateprotamine membrane. Tissue Eng, 2005; 11: 709-14.
(3) Simsek O, Arikan S. Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro. Anim Reprod, 2015; 12: 931-38
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