Effects of cyclic adenosine monophosphate modulators on maturation and quality of vitrified-warmed germinal vesicle stage mouse oocytes.

BACKGROUND: It is still one of the unresolved issues if germinal vesicle stage (GV) oocytes can be successfully cryopreserved for fertility preservation and matured in vitro without damage after warming. Several studies have reported that the addition of cyclic adenosine monophosphate (cAMP) modulators to in vitro maturation (IVM) media improved the developmental potency of mature oocytes though vitrification itself provokes cAMP depletion. We evaluated whether the addition of cAMP modulators after GV oocytes retrieval before vitrification enhances maturation and developmental capability after warming of GV oocytes. METHODS: Retrieved GV oocytes of mice were divided into cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). Then, GV oocytes were cultured with or without dibutyryl-cAMP (dbcAMP, cAMP analog) and 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) during the pre-vitrification period for 30 min. RESULTS: One hour after warming, the ratio of oocytes that stayed in the intact GV stage was significantly higher in groups treated with cAMP modulators. After 18 h of IVM, the percentage of maturation was significantly higher in the COC group treated with dbcAMP. The expression of F-actin, which is involved in meiotic spindle migration and chromosomal translocation, is likewise increased in this group. However, there was no difference in chromosome and spindle organization integrity or developmental competence between the MII oocytes of all groups. CONCLUSIONS: Increasing the intracellular cAMP level before vitrification of the GV oocytes maintained the cell cycle arrest, and this process may facilitate oocyte maturation after IVM by preventing cryodamage and synchronizing maturation between nuclear and cytoplasmic components. The role of cumulus cells seems to be essential for this mechanism.

Effects of antifreeze proteins on the vitrification of mouse oocytes: comparison of three different antifreeze proteins.

STUDY QUESTION: Can antifreeze proteins (AFPs) from three different sources improve the efficacy of mouse oocyte vitrification? SUMMARY ANSWER: Treatment with AFPs can improve both murine oocyte quality and embryo development, and reduce reactive oxygen species (ROS) production in vitrified-warmed oocytes. WHAT IS KNOWN ALREADY: A previous study discovered that vitrification of immature oocytes and 2-cell stage embryos of mice augmented with antifreeze glycoproteins at 40 mg/ml dramatically improved the morphological integrity of the samples, suggesting that AFPs have the ability to inhibit ice formation and stabilize the plasma membrane. STUDY DESIGN, SIZE, DURATION: Metaphase II oocytes were obtained from 4-week-old BD-F1 mice. AFPs from bacteria (Flavobacterium frigoris ice-binding protein (FfIBP)), yeast (Glaciozyma sp. ice-binding protein (LeIBP)) and fish (Type III AFP) were added to the vitrification and warming solutions individually. Survival and development, meiotic spindle organization, intracellular ROS, mitochondrial activity, DNA double-strand breaks (DSBs) and repair of damaged DNA were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Vitrification of oocytes was performed with the CryoTop (equilibration solution: 7.5% ethylene glycol (EG) and 7.5% 1,2-propandiol (PROH) for 5 min; vitrification solution: 15% EG, 15% PROH and 0.5 M sucrose for 1 min). Warming was performed in three steps with decreasing concentrations of sucrose (1.0, 0.5 and 0.25 M sucrose). MAIN RESULTS AND THE ROLE OF CHANCE: AFP treatment can improve murine oocyte quality and embryo development. Survival rates, cleavage rates and blastocyst rates (blastocyst per cleaved and per survived oocytes) of oocytes in AFP-treated groups were significantly higher than those in the control group [75.0, 89.0, 90.0 and 85.0% for survival rate (P = 0.012); 58.7, 89.0, 87.8 and 81.2% for cleavage rate (P = 0.003); 52.3, 87.7, 78.5 and 76.8% for blastocyst per cleaved oocytes (P < 0.01); 30.7, 78.0, 68.9 and 62.4% for blastocyst per survived oocytes (P < 0.01) in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively]. The mean (±SD) number of apoptotic blastomeres per blastocyst was significantly lower in AFP-treated groups than in the control group (9.1 ± 1.0, 2.0 ± 1.7, 2.3 ± 1.2 and 2.7 ± 2.4 in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P = 0.040). FfIBP treatment was the most effective in maintaining normal meiotic spindle organization and chromosome alignment (52.0, 92.0, 80.0 and 83.0% in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P < 0.01). Intracellular ROS levels (mean ± SD) significantly decreased in the AFP-treated groups (17.0 ± 11.2, 8.4 ± 8.2, 10.3 ± 6.4 and 11.6 ± 12.3 in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P < 0.01), and the FfIBP and LeIBP groups had significantly lower DNA DSBs, compared with controls (65.2, 30.8, 44.4 and 55.8% in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The origins of FfIBP and LeIBP were bacteria and yeast, respectively. Therefore, treatment of human oocytes and embryos with these AFPs should be tested before clinical application. WIDER IMPLICATIONS OF THE FINDINGS: After further research, AFPs can potentially be applied to human oocyte cryopreservation to improve the efficacy of vitrification. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). The authors have no conflict of interest to declare.

Inhibition of the ubiquitin-proteasome system leads to delay of the onset of ZGA gene expression.

In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos.

Deposition of acetylated histones by RNAP II promoter clearance may occur at onset of zygotic gene activation in preimplantation mouse embryos.

We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA). Using immunofluorescence staining, we observed that the nuclear localization of RNAP II was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi. In a transient gene expression assay using a plasmid reporter gene (pß-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+ gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for at least 4 hours after injection. We found that the methylation status in the chicken ß-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA. Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo. Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state. Taken together, these results suggest that deposition of selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo.

Assessment of vascular endothelial growth factor expression and apoptosis in the ovarian graft: can exogenous gonadotropin promote angiogenesis after ovarian transplantation?

OBJECTIVE: To evaluate the effects of gonadotropin on angiogenesis by assessing vascular endothelial growth factor (VEGF) expression in rat ovaries transplanted after freezing and thawing. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. ANIMAL(S): Sixty immature female rats. INTERVENTION(S): Frozen-thawed ovaries were autotransplanted into the SC tissue of 60 rats (ages between 21 and 28 days). After transplantation, either pregnant mare's serum gonadotropin (PMSG) or saline was administered. The grafted ovaries were collected 2, 7, and 30 days after transplantation for evaluation. MAIN OUTCOME MEASURE(S): Assessment of the morphology and number of follicles, evaluation of apoptosis, and analysis of VEGF expression in the grafted ovaries. RESULT(S): Most follicles in the grafts were apoptotic on day 2 but recovered by day 7. The proportion of antral follicles and corpora lutea in the graft correlated with the duration after transplantation. A significant increase in the expression of VEGF188 mRNA was noticed in the grafted ovaries on day 2. Moreover, the mRNA expression in the PMSG group was higher than that in the control group. The increased VEGF protein production in the graft was confirmed by Western blot analysis. CONCLUSION(S): In ovariectomized animals, gonadotropin treatment may not provide any added benefits for folliculogenesis and angiogenesis. Nevertheless, a significant increase in the VEGF188 isoform in the gonadotropin-treated group may suggest the positive effect of exogenous gonadotropin therapy in the early stages of angiogenesis.

Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation.

BACKGROUND: Previous studies showed that immature oocytes stored in ovarian tissue could develop to the mature stage after transplantation. However, the quality and competency of the oocytes developed in xenografted ovarian tissue have never been investigated. As a pilot study to investigate this uncharted issue, we evaluated microtubule organization and chromatin configuration of human oocytes harvested from xenografted frozen-thawed ovarian tissue. METHODS: Frozen-thawed human ovarian tissue was transplanted into severe combined immunodeficient mice. All animals were stimulated with gonadotrophin from 20 weeks after transplantation. Grafts were recovered 36 h after hCG administration. The oocytes were retrieved from the antral follicles (>2 mm diameter), cultured in vitro, stained for microtubule and chromatin localization. RESULTS: Five oocytes from 21 female mice and seven oocytes from nine male mice were retrieved. Immunocytochemical examinations of these oocytes after in vitro maturation revealed only two developed to the metaphase II stage. Most oocytes were between prophase and metaphase with abnormal microtubule organization and chromatin configuration. CONCLUSIONS: Immature oocytes in stored human ovarian tissue can grow to maturity in host animals after xenotransplantation. Retrieval of oocytes from the xenograft can be carried out and is reproducible. However, many oocytes, grown in host animals and further matured in vitro, showed aberrant microtubule organization and chromatin patterns.