Methodological approaches for vitrification of bovine oocytes

Zygote ◽  
2020 ◽  
pp. 1-11
Author(s):  
Linda Dujíčková ◽  
Alexander V. Makarevich ◽  
Lucia Olexiková ◽  
Elena Kubovičová ◽  
František Strejček
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Oocyte Vitrification ◽  
Factors Affecting ◽  
Oocyte Stage ◽  
Follicle Size ◽  
Oocyte Donors ◽  
Methodological Approaches ◽  
Bovine Oocytes

Summary 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.

scholarly journals Effects of C-type natriuretic peptide on meiotic arrest and developmental competence of bovine oocyte derived from small and medium follicles

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhenwei Jia ◽  
Xueli Wang
Keyword(s):  
Natriuretic Peptide ◽  
Basal Medium ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
The Novel ◽  
Bovine Oocyte ◽  
Meiotic Arrest ◽  
Follicle Size

Abstract The present study aimed to evaluate the effects of C-type natriuretic peptide (CNP) on meiotic arrest and developmental competence of bovine oocyte derived from follicles of different sizes. Collected immature cumulus-oocyte complexes from small follicles (< 3 mm) and medium follicles (3–8 mm) were cultured for 6 h in basal medium supplementated without or with 200 nM CNP. We observed that CNP effectively sustained meiotic arrest at germinal vesicle stage in in vitro cultured bovine oocytes from follicles of different sizes. Moreover, CNP treatment significantly improved the levels of cGMP in both cumulus cells and oocytes, as well as the levels of cAMP in oocytes regardless of follicle size. Based on the above results, we tested the effect of a novel in vitro maturation (IVM) system based on CNP-pretreatment, including a pre-IVM phase for 6 h using 200 nM CNP, followed by a extended IVM phase for 28 h, on developmental competence of bovine oocyte derived from small follicles (< 3 mm) and medium follicles (3–8 mm) compared to standard IVM system. The results showed that athough the novel IVM system based on CNP-pretreatment enhanced the developmental potencial of oocytes obtained from large follicles, but had no effect on the developmental comptence of oocytes obtained from small follicles.

Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size

2003 ◽  
Vol 15 (4) ◽  
pp. 223 ◽  
Author(s):  
Muhammad Rizwan Yousaf ◽  
Kazim Raza Chohan
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Morphology ◽  
Vitro Fertilization ◽  
Size Dependent ◽  
Size Groups ◽  
Follicle Size ◽  
Fetal Bovine ◽  
Meiotic Competence

The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus–oocyte complexes (COCs) were collected from 1–<2, 2–<3, 3–<4, 4–<6 and 6–<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL−1 hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 μg mL−1 follicle-stimulating hormone, 10 μg mL−1 luteinizing hormone, 1.5 μg mL−1 oestradiol, 75 μg mL−1 streptomycin, 100 IU mL−1 penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1–<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1–<2 mm (107.7 ± 1.6 μm), 2–<3 mm (108 ± 1.1 μm) and 3–<4 mm (114.6 ± 1.3 μm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4–<6 mm (124.4 ± 1.3 μm) and 6–<8 mm (131.9 ± 1.4 μm) in diameter. A majority of oocytes (P < 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4–<6- and 6–<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2–<3- and 3–<4-mm follicles, respectively, whereas significantly (P < 0.05) more oocytes from 4–<6- and 6–<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.

Dynamic changes in the global transcriptome of bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

2018 ◽  
Vol 30 (10) ◽  
pp. 1298 ◽  
Author(s):  
Jianwei Huang ◽  
YongShun Ma ◽  
Shao Wei ◽  
Bo Pan ◽  
Yu Qi ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Oocyte Vitrification ◽  
Kegg Pathway Database ◽  
Global Transcriptome ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
Regulation Of Actin Cytoskeleton ◽  
Mrna Transcriptome

This study was conducted to investigate the effect of vitrification on the dynamics of the global transcriptome in bovine germinal vesicle (GV) oocytes and their in vitro-derived metaphase II (MII) oocytes. The GV oocytes were vitrified using the open-pulled straw method. After warming, GV oocytes and the resulting MII-stage oocytes were cultured in vitro for 2 h and 24 h respectively and were then collected. The fresh GV oocytes and their in vitro-derived MII oocytes were used as controls. Then, each pool (fresh GV, n = 3; vitrified GV, n = 4; fresh MII, n = 1 and MII derived from vitrified GV, n = 2) from the different stages was used for mRNA transcriptome sequencing. The results showed that the in vitro maturation rates of GV oocytes were significantly decreased (32.36% vs 53.14%) after vitrification. Bovine GV oocyte vitrification leads to 12 significantly upregulated and 19 downregulated genes. After culturing in vitro, the vitrification-derived MII oocytes showed 47 significantly upregulated and six downregulated genes when compared with those from fresh GV oocytes. Based on molecular function–gene ontology terms analysis and the Kyoto encyclopaedia of genes (KEGG) pathway database, the differentially expressed genes were associated with the pathways of cell differentiation and mitosis, transcription regulation, regulation of actin cytoskeleton, apoptosis and so on, which potentially result in the lower in vitro development of GV bovine oocytes.

Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

2009 ◽  
Vol 21 (5) ◽  
pp. 655 ◽  
Author(s):  
Ester Siqueira Caixeta ◽  
Paula Ripamonte ◽  
Maurício Machaim Franco ◽  
José Buratini Junior ◽  
Margot Alves Nunes Dode
Keyword(s):  
Gene Expression ◽  
Bos Indicus ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Marker Genes ◽  
Oocyte Competence ◽  
Follicular Size ◽  
Follicle Size

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0–3.0, 3.1–6.0, 6.1–8.0 and ≥8.1 mm. Cumulus–oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription–polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles >6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P < 0.05) in oocytes from follicles ≥8.1 mm in diameter than in oocytes from follicles <6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

scholarly journals 288 RELATIONSHIP BETWEEN CHROMATIN ORGANIZATION AND OOCYTE - CUMULUS CELL COMMUNICATION IN GERMINAL VESICLE STAGE BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 294
Author(s):  
V. Lodde ◽  
C. Galbusera ◽  
S. Modina ◽  
M.S. Beretta ◽  
A. Lauria ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Hoechst 33342 ◽  
Oocyte Growth ◽  
The Status ◽  
Chromatin Configuration ◽  
Bovine Oocytes

Chromatin configuration in the germinal vesicle (GV) undergoes dynamic changes during oocyte growth, and the progressive chromatin condensation has been related to the acquisition of embryonic developmental potential. However, little is known about the mechanisms that regulate chromatin remodeling. In immature mouse oocytes, chromatin condensation and redistribution around the nucleolus are associated with transcriptional repression in both in vivo-derived and in vitro-cultured oocytes in the presence of an intact cumulus oophorus (de la Fuente et al. 2001 Dev. Biol. 229, 224). It is widely accepted that oocyte communication with the somatic cell compartment is essential for both oocyte growth and acquisition of meiotic competence (Eppig et al. 1997 Hum. Reprod. 12, 127). In particular, cumulus cells play an active role in modulating the levels of transcription in the nucleoplasm and in perinuclear domains as well as in chromatin configuration of GV stage oocytes. In cattle, a heterogeneous population of cumulus-oocyte complexes (COCs) has been found after isolation from the follicle, and this is characterized by a different functional degree of gap junction-mediated communication (Luciano et al. 2004 Biol. Reprod. 70, 465). This study was aimed at investigating the possible correlation between the chromatin configuration of immature bovine oocytes and the status of communication between the oocyte and cumulus cells, and oocyte developmental competence. In the first experiment, 138 COCs, isolated from follicles 2–6 mm in diameter, were injected with a 3% solution of Lucifer Yellow to assess the communication status between oocytes and cumulus cells. Successively, COCs were freed of cells, and denuded oocytes (DOs) were stained with Hoechst 33342 to determine the chromatin configuration. In a second experiment, 330 COCs were denuded and stained with Hoechst 33342 in order to assess chromatin configuration and then matured in vitro according to their GV stage. After IVM, DOs were fertilized, and presumptive zygotes were cultured for 7 days at which time blastocyst rate was assessed. Data were analyzed by ANOVA and Fisher's PLSD test. Three stages of GV oocytes were identified: GVI, with filamentous chromatin distributed in the nucleoplasm; GVII, with chromatin condensed into thick clumps; and GVIII, with chromatin condensed into a single clump. The GVIII stage showed a lower proportion of functional open communication than the GVI and GVII groups (8.5 vs. 45.7 and 46.1, respectively, P < 0.05). However, when compared with each other, the GVI stage oocytes showed lower embryonic developmental competence (12.9 in GVI vs. 22.1 and 24.2 in GVII and GVIII, respectively, P < 0.05). Our findings indicate that the status of communication between oocytes and cumulus cells could be related to the chromatin organization in immature bovine oocytes. A direct correlation between the communications grade, the modulation of oocyte transcriptional activity, and the acquisition of oocyte developmental competence remain to be confirmed. This work was supported by a 2003 UniMi Grant.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Shiori Ashibe ◽  
Kanade Irisawa ◽  
Ken Yokawa ◽  
Yoshikazu Nagao
Keyword(s):  
Treatment Group ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Free Calcium ◽  
Parthenogenetic Development ◽  
Early Embryos ◽  
Bovine Oocytes

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence

2021 ◽  
Author(s):  
Dulama Richani ◽  
Robert B Gilchrist
Keyword(s):  
Protein Synthesis ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Protein Synthesis Inhibitors ◽  
Meiotic Arrest ◽  
Oocyte Developmental Competence

Abstract Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents which increase intra-oocyte cAMP or prevent its degradation have been predominantly used, however agents such as kinase and protein synthesis inhibitors have also been trialled. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

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