Factors affecting cryotolerance of mammalian oocytes.

Cryopreservation of oocytes is an important tool for preserving genetic resources and for farm animals breeding. Processes taking place during vitrification affect oocytes and result in their reduced developmental capacity and lower fertilisation rates of cryopreserved oocytes. Further improvement in cryopreservation techniques is still required. Several authors already summarized the actual state and perspectives of oocyte cryopreservation as well as potential approaches to improve their development after thawing. The aim of this review is to specify factors affecting cryotolerance of mammalian oocytes, especially bovine in vitro matured oocytes, and to identify the areas, where more efforts were made to improve the success of oocyte cryopreservation. These factors include oocyte lipid content, membrane composition, mRNA protection, cytoskeleton stabilization and application of such potential stimulators of cell cryotolerance as antioxidants, growth factors or antifreeze proteins.

Distribution of tetraspanins in bovine ovarian tissue and fresh/vitrified oocytes.

Tetraspanin proteins are mostly known as organizers of molecular complexes on cell membranes, widely expressed on the surface of most nucleated cells. Although tetraspanins participate in many physiological processes of mammals, including reproduction, their relevance to the processes of folliculogenesis and oogenesis has not yet been fully elucidated. We bring new information regarding the distribution of tetraspanins CD9, CD81, CD151, CD82, and CD63 at different stages of follicular development in cattle. The found distribution of tetraspanin CD9, CD63, and integrin alpha V in similar areas of ovarian tissue outlined their possible cooperation. We also describe yet-unknown distribution patterns of CD151, CD82, and CD63 on immature and mature bovine oocytes. The unique localization of tetraspanins CD63 and CD82 in the zona pellucida of bovine oocytes suggested their involvement in transzonal projections. Furthermore, we present an unchanged distribution pattern of the studied tetraspanins in vitrified mature bovine oocytes. The immunofluorescent analysis was supplemented by in silico data addressing tetraspanins expression in the ovarian cells and oocytes across several species. The obtained results suggest that in the study of the oocyte development and potentially the fertilization process of cattle, the role of tetraspanins and integrins should also be taken into account.

The morphological and functional ultrastructure of cells in pre-implantation embryos.

Cells of pre-implantation embryos are equipped with many morphological and functional systems through which they can synthesize specific proteins and effectively ensure the protection of early embryonic development. Here we present evidence for the existence of these systems in morphologically normal and abnormal bovine blastocyst stage embryos in vivo at the ultrastructural and actin cytoskeleton levels. The appearance of organelles in the trophectoderm (TE) and inner cell mass (ICM) cells, responsible for their synthetic activities and their role in the development of early bovine embryos are described. We point out the importance of endocytic processes and the participation of extracellular vesicles in the formation of intercellular contacts and homeostasis of the embryo microenvironment. Several changes in the ultrastructural morphology of embryos produced by different methods (ICSI, parthenogenetic AC/DC electrical activation, IVF with separated sperm) and freezing/thawed embryos are described. We also show alterations occurred in the organelles after viral contamination of embryos with BHV-1 and BVDV viruses, and in embryos from over-conditioned cows. Recorded changes in organelles and appearance of cellular autophagic structures (vesicles, multivesicular bodies and autophagolysosomes) may negatively affect embryo metabolism and lead to the emergence of pathological processes in TE and ICM cells of preimplantation embryos.

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

Methodological approaches for vitrification of bovine oocytes.

Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

Astaxanthin Added during Post-Warm Recovery Mitigated Oxidative Stress in Bovine Vitrified Oocytes and Improved Quality of Resulting Blastocysts.

Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was to find whether astaxanthin (AX), a xanthophyll carotenoid, could mitigate deteriorations that occurred during the vitrification/warming process in bovine oocytes. Astaxanthin (2.5 µM) was added to the maturation medium during the post-warm recovery period of vitrified oocytes for 3 h. Afterward, the oocytes were fertilized in vitro using frozen bull semen and presumptive zygotes were cultured in the B2 Menezo medium in a co-culture with BRL-1 cells at 38.5 °C and 5% CO2 until the blastocyst stage. AX addition significantly reduced ROS formation, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity in vitrified oocytes. Although the effect of AX on embryo development was not observed, it stimulated cell proliferation in the blastocysts derived from vitrified oocytes and improved their quality by upregulation or downregulation of some genes related to apoptosis (BCL2, CAS9), oxidative stress (GPX4, CDX2), and development (GJB5) compared to the vitrified group without AX. Therefore, the antioxidant properties of astaxanthin even during short exposure to bovine vitrified/warmed oocytes resulted in improved blastocyst quality comparable to those from fresh oocytes.

Glutathione during Post-Thaw Recovery Culture Can Mitigate Deleterious Impact of Vitrification on Bovine Oocytes.

Vitrification of bovine oocytes can impair subsequent embryo development mostly due to elevated oxidative stress. This study was aimed at examining whether glutathione, a known antioxidant, can improve further embryo development when added to devitrified oocytes for a short recovery period. Bovine in vitro matured oocytes were vitrified using an ultra-rapid cooling technique on electron microscopy grids. Following warming, the oocytes were incubated in the recovery medium containing glutathione (0, 1.5, or 5 mmol L-1) for 3 h (post-warm recovery). Afterwards, the oocytes were lysed for measuring the total antioxidant capacity (TAC), activity of peroxidase, catalase and glutathione reductase, and ROS formation. The impact of vitrification on mitochondrial and lysosomal activities was also examined. Since glutathione, added at 5 mmol L-1, significantly increased the TAC of warmed oocytes, in the next set of experiments this dose was applied for post-warm recovery of oocytes used for IVF. Glutathione in the recovery culture did not change the total blastocyst rate, while increased the proportion of faster developing blastocysts (Day 6-7), reduced the apoptotic cell ratio and reversed the harmful impact of vitrification on the actin cytoskeleton. These results suggest that even a short recovery culture with antioxidant(s) can improve the development of bovine devitrified oocytes.

The Cryopreserved Sperm Traits of Various Ram Breeds: Towards Biodiversity Conservation.

The aim of our research was to compare three Slovak sheep breeds in the quality parameters of cryopreserved sperm. The ejaculates of Slovak Dairy (SD), Native Wallachian (NW), and Improved Wallachian (IW) sheep rams (n = 12) were collected by electro-ejaculation. Heterospermic samples were created from suitable ejaculates, separately for each breed (at least 90% of total and 80% of progressive motility). Samples were equilibrated in a Triladyl® diluent and frozen by automated freezing. Sperm samples were subjected to the motility, morphology, (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), fertilizing capability (penetration/fertilization test (P/F) in vitro) and acrosomal status (transmission electron microscopy) assays before freezing and after thawing. It was found that there were no significant differences (p < 0.05) between the evaluated breeds in motility, viability, apoptosis, morphological properties, and fertilizing ability of cryopreserved sperm. Significant differences occurred in acrosomal status. Our results demonstrate that the use of the selected cryopreservation protocol is suitable for at least three different sheep breeds, which can greatly benefit the biodiversity protection and simplifies the creation of an animal genetic resources gene bank.

Development and ultrastructure of bovine matured oocytes vitrified using electron microscopy grids.

The aim of this study was to establish a methodology of cryopreservation of cattle oocytes and the quality assessment of oocytes and subsequent embryos produced in vitro under our laboratory conditions. Previously in vitro matured (IVM) oocytes were vitrified in minimum volume by ultra-rapid cooling technique. The oocytes were put into the equilibration solution (3% ethylene glycol in M199-HEPES + 10% foetal bovine serum) for 12 min, transferred to vitrification solution (30% ethylene glycol + 1 M sucrose in M199-HEPES + 10% foetal bovine serum) at room temperature for 25 s, then placed onto nickel electron microscopy grids and plunged into liquid nitrogen. After warming 75% of the oocytes were assessed as viable. Part of viable oocytes was taken for electron microscopy, the remaining oocytes were fertilized in vitro, and the presumptive zygotes were cultured until the blastocyst stage. Embryo cleavage and blastocyst rates in vitrified group after warming were 64.98% and 17.3%, resp. versus 70.72% and 25.54% in the control group (P < 0.05). No significant differences were found in the blastocyst total cell number, TUNEL and dead cell indexes between both groups. Ultrastructure of vitrified oocytes showed damages in smooth endoplasmic reticulum (SER) vesicles and lipid droplets as well as irregular arrangement of solitary cortical granules. Several mitochondria were damaged and the microtubules around the chromosomes were less occurred compared to the control group. However, the extent of injuries was lower than reported by other authors studying the ultrastructure of vitrified bovine oocytes, what is also supported by the better development of our oocytes after IVF. In conclusion, the designed oocyte vitrification technique ensures obtaining the blastocysts of the quality comparable to the fresh oocytes.