4 SUPPRESSION OF ASH2L ALTERS DNA METHYLATION AND HISTONE PATTERNS DURING BOVINE EMBRYONIC DEVELOPMENT

Epigenetic patterns established during early bovine embryogenesis via DNA methylation and histone modification patterns are essential for proper gene expression and embryonic development. We have previously discovered that suppression of absent, small, or homeotic-like (ASH2L) with small interfering RNA (siRNA) had no significant effect during in vitro embryo development when compared with its respective control (31.3 ± 2.0% standard error of the mean, n = 466 v. 34.8 ± 1.9%, n = 418). Analysing DNA methylation and histone modifications via immunocytochemistry will further explain the role of ASH2L during embryonic development, specifically at the blastocyst stage. In this experiment, we obtained mature bovine oocytes from a commercial supplier (De Soto Biosciences, Seymour, TN) and preformed IVF following standard laboratory protocol. Eighteen hours after IVF, presumptive zygotes were divided into 3 treatments: noninjected controls, nontargeting siRNA injected controls (siNULL), and injection with siRNA targeting ASH2L (siASH2L). Each embryo was injected with ~100 pL of 20 nM siRNA previously verified to suppress expression of ASH2L by ~79%. Embryos were cultured in Bovine Evolve (Zenith Biotech, Guilford, CT) supplemented with 4 mg mL–1 of BSA (Probumin, Millipore) for 7 days. Blastocysts from each treatment (N = 601) were fixed and prepared for immunocytochemistry following standard laboratory protocol. The following primary antibodies were used to target specific DNA and histone methylation marks: 5mc mAb (Epigentek, Farmingdale, NY), 5hmc pAb, H3K4me3 pAb (Active Motif, Carlsbad, CA), H3K4me2 pAb, H3K9me2–3 mAb, and H3K27me3 mAb (Abcam, Cambridge, MA). Embryos were fluorescently labelled with the following secondary antibodies: Alexa Flour 488 Goat Anti-Rabbit, Alexa 488 Donkey Anti-Goat, and Alexa Flour 594 Goat Anti-Mouse (Invitrogen, Carlsbad, CA). The DNA was stained with Hoechst 33342 (Invitrogen). Fluorescent images were captured using the Zeiss Stallion digital imaging work station. Ratio averages (targeting mark/DNA) were calculated and statistical analysis performed using one-way ANOVA and Tukey’s honestly significant difference to assess treatment effects. The ratio of DNA methylation to total DNA increased in siASH2L as compared with control and siNULL embryos (0.35 ± 0.01, 0.26 ± 0.02, and 0.30 ± 0.01, respectively; P < 0.01). The 5hmC was inversely related to 5mC levels and decreased in siASH2L embryos (0.75 ± 0.01, 0.93 ± 0.02, 0.87 ± 0.02, respectively; P < 0.0001). The H3K4me3 and H3K27me3 are also inversely related with decreased H3K4me3 in siASH2L versus control and siNULL embryos (0.48 ± 0.02, 0.57 ± 0.02, 0.58 ± 0.02, respectively; P < 0.001) and increased H3K27me3 (0.62 ± 0.02, 0.053 ± 0.01, 0.54 ± 0.02, respectively; P < 0.001). No differences were observed in H3K9me2–3 or H3K4me2 labelling across treatments. These results indicate that ASH2L may play a role in DNA methylation by decreasing 5mc and 5hmc conversion, which is a key event during early embryonic development. Suppression of ASH2L also alters global levels of H3H4me3 and H3K27me3, which may lead to transcription aberrations. Further analysis of siASH2L embryos via RNA-seq will help define its role during early embryonic development.

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232 EFFECT OF DIETARY FAT SUPPLEMENTATION ON OOCYTE AND EMBRYO QUALITY AND ON EARLY EMBRYONIC DEVELOPMENT OF SHEEP

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab232 ◽

2006 ◽

Vol 18 (2) ◽

pp. 224

Author(s):

G. Cancino-Arroyo ◽

R. Ake-López ◽

J. Herrera ◽

F. Centurion ◽

A. Ordoñez-León

Keyword(s):

Embryonic Development ◽

Corn Oil ◽

Body Condition Score ◽

Prostaglandin F2α ◽

Oocyte Quality ◽

Early Embryonic Development ◽

Control Group ◽

Fat Supplementation ◽

Significant Difference

The objective was to evaluate the effect of fat supplementation on oocyte quality and in vitro embryonic development (48 h). A total of 18 ewes, with a body condition score of 2.5 to 3 points out of 5, having had three to four lamb births, and at three to four months post-lamb birth, were distributed between an experimental oil group (OG; n = 9) that received corn oil (4% of the MS/diet) and a control group (CG; n = 9) that didn't receive oil. The two groups were maintained in confinement for 21 days (the duration of the experiment) and fed first with a concentrate diet followed by forage. The animals had access to minerals and water ad libitum. The diets were similar in energy (10.3 ± 0.05 MJ/s/d) and protein (141.75 ± 5.7 gPC/s/d) for both groups. The estrous cycle was synchronized (14 days) with intravaginal sponges (40 mg of fluorogestone acetate), inserted 7 days after the beginning of the diets. The end of the diet coincided with the retirement of the sponges. One day before sponge retirement, 75 mg prostaglandin F2α per sheep was administered, followed by ovarian stimulation with 1000 IU of pregnant mare serum gonadotropin (PMSG). Follicular diameter was determined by ventral laparotomy with the aid of a micrometer. Follicles were classified as small (2 to 2.9 mm), medium (3 to 4.9 mm), and large (>5 mm); oocytes were collected in TCM-199 medium. Oocytes were classified as excellent, good, fair, or low quality and transferred to Petri dishes in drops (50 mL) of TCM-199. Oocytes were matured and fertilized in vitro and cultured for 48 h. Oocyte quality as well as maturation, fertilization, and cleavage rates were compared by ANOVA. Ewes from the OG group presented a statistically higher proportion of oocytes with excellent quality (42%; P < 0.05) than GT ewes (26%). The proportion of good quality and fair quality oocytes was similar among groups (P > 0.05). A higher proportion of oocytes of low quality was found in the control group than in the OG group (40% vs. 18%); however, there was no significant difference (P > 0.05). Higher rates of maturation, fertilization, and early development were found in the OG compared with the CG (81.8, 60.6, and 36.4 vs. 68.6, 42.9, and 17.1, respectively); however, the differences were not significant (P > 0.05). In conclusion, the addition of 4% corn oil in the diet improved the quality of the oocytes; however, it had no significant effect on early embryonic development.

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Fertilization and early embryonic development of in vitro matured metaphase I oocytes in patients with unexpected low oocyte maturity rate

Zygote ◽

10.1017/s0967199421000654 ◽

2021 ◽

pp. 1-5

Author(s):

Nafiye Yılmaz ◽

Şebnem Özyer ◽

Derya Taş ◽

Mehmet Caner Özer ◽

Ayten Türkkanı ◽

...

Keyword(s):

Embryonic Development ◽

Fertilization Rate ◽

Early Embryonic Development ◽

Oocyte Maturity ◽

Significant Difference ◽

Maturation Rate ◽

Developmental Capacity ◽

Metaphase I

Summary To determine the fertilization and embryonic potential of immature metaphase I (MI) oocytes in patients with low oocyte maturity rate in whom the percentage of mature oocytes obtained was less than 75% of the total retrieved ones. In vivo matured metaphase II (MII) oocytes (MII-ICSI, n = 244), and in vitro matured MI oocytes (MI-MII-ICSI, n = 202) underwent an intracytoplasmic sperm injection (ICSI) procedure. Maturation rate, fertilization rate and early embryonic development were compared in both groups. In total, 683 oocytes were collected from 117 ICSI cycles of 117 patients. Among them, 244 (35.7%) were mature MII and 259 (37.9%) were MI after the denudation process. Of those 259 MI oocytes, 202 (77.9%) progressed to MII oocytes after an incubation period of 18–24 h. The maturation rate was 77.9%. Fertilization rate was found to be significantly higher in the rescued in vitro matured MI oocyte group when compared with the in vivo matured MII oocyte group (41.6% vs 25.8%; P = 0.0006). However, no significant difference was observed in terms of cleavage rates on days 2 and 3 between the groups (P = 0.9126 and P = 0.5031, respectively). There may be unidentified in vivo factors on the oocyte maturation causing low developmental capacity in spite of high fertilization rates in the group of patients with low oocyte maturity rate. Furthermore, studies are needed to determine the appropriate culture characteristics as well as culture period and ICSI timing of these oocytes.

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100 SEQUENTIAL VERSUS SINGLE-STEP MEDIUM FOR RHESUS MACAQUE EMBRYO CULTURE

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab100 ◽

2016 ◽

Vol 28 (2) ◽

pp. 180

Keyword(s):

Embryonic Development ◽

Embryo Culture ◽

Culture Media ◽

Blastocyst Stage ◽

Single Step ◽

Blastocyst Development ◽

Equal Variance ◽

Significant Difference ◽

Health Studies

The nonhuman primate (NHP) is a valuable translational model for human health studies and is widely used to investigate pre-implantation embryo development. Central to these investigations is the dependency on in vitro embryo culture (IVC). Since 2001, the single-step hamster embryo culture medium (HECM) has been the accepted standard for NHP IVC. With recent advances in formula optimization for IVC in human clinics, a re-examination of optimal NHP IVC media is warranted. Thus, two types of commercially available IVC media routinely used in human applications were compared with HECM-9: Global (single-step; LifeGlobal Group, Guilford, CT, USA), and Quinns Advantage (sequential; SAGE, Trumbull, CT, USA). Normally cycling, adult rhesus monkeys (n = 3) underwent controlled ovarian stimulations, and follicles were aspirated via laparoscope. Recovered ova were fertilized in vitro and the resultant zygotes (n = 138) were cultured for 9 days in HECM-9, Global, or Quinns with 10% protein supplement at 37.5°C in humidified tri-gas (6% CO2, 5% O2, and 89% N). Single-step media (HECM-9 and Global) were refreshed every two days while embryos were cultured for Days 1–3 in Quinns Advantage Cleavage medium without being replaced and in Quinns Advantage Blastocyst medium for Days 4–9 with medium changes every 2 days. Embryos were observed for cleavage, compaction, and blastocyst development. Proportional data with equal variance and normal distribution were analysed by one-way ANOVA, and significance was determined post-hoc by the Holm-Sidak method with P < 0.05. Developmental stage data ± s.e.M are presented in Table 1; a change in superscript indicates a significant difference within the column. There was no difference in embryonic cleavage or morula compaction between the three culture media evaluated, indicating no obvious differences in their effects on embryonic development 1 to 3 days after fertilization. However, a greater proportion of blastocysts developed in Global medium compared with HECM-9, and though it was not statistically different, embryos cultured in Global tended to reach the blastocyst stage more frequently than those in Quinns. Although not significant due to large variances in each group, blastocyst expansion also tended to occur more frequently in Global medium than in HECM-9 or Quinns. Taken together, these data indicate that single-step Global is as supportive of early embryonic development as HECM-9 but is better formulated to facilitate later stage differentiation and would be better suited for use in updated standard NHP IVC protocols. Table 1.Cleavage, compaction, blastocysts, and expansion of embryos in HECM-9, Global, and Quinns media

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299 IN VITRO DEVELOPMENT OF IMMATURE PORCINE OOCYTES FERTILIZED IN VITRO TO THE BLASTOCYST STAGE

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab299 ◽

2005 ◽

Vol 17 (2) ◽

pp. 300

Author(s):

T. Somfai ◽

K. Kikuchi ◽

S.Y. Medvedev ◽

A. Onishi ◽

M. Iwamoto ◽

...

Keyword(s):

Embryonic Development ◽

Inner Cell Mass ◽

Polar Body ◽

Cell Mass ◽

Cell Number ◽

Blastocyst Stage ◽

Oocyte Activation ◽

Cleavage Rate ◽

Significant Difference

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in this study. After in vitro maturation (IVM) for 48 h of cumulus-oocyte complexes, 75.4% (n = 442) of them extruded a visible polar body (PB). Most oocytes with a polar body (PB+ group) were found to be at metaphase II (M-II) stage (91.4%). Most oocytes without a visible polar body (PB− group, n = 144) appeared to be arrested at the germinal vesicle (GV) (41.6%) and first meiotic metaphase (M-I) (34.0%) stages. After IVF of oocytes (the day of IVF = Day 0), there was no significant difference between PB+ and PB− groups in rates of sperm penetration, monospermy, and oocyte activation after the penetration. Embryonic development was assessed by staining with 1% orcein. On Day 2, although there was no difference between the embryo cleavage in PB+ (n = 447) and PB− (n = 217) groups (47.0% and 35.9%, respectively), PB+ embryos had more cells than the PB− embryos (3.37 and 2.81 cells, respectively) (P < 0.05; ANOVA). On Day 4, the cleavage rate of PB+ embryos was higher than that of PB− embryos (45.4% and 24.3%, respectively), and PB+ embryos had more cells than the PB− embryos (8.26 and 6.0 cells, respectively) (P < 0.05; ANOVA). On Day 6, a significantly higher number of PB+ embryos developed to the blastocyst stage than that of the PB− embryos (34.6% and 20.7%, respectively) (P < 0.05). However, by subtracting the GV oocytes from the PB− group, there was no difference in blastocyst rates between the M-I arrested and M-II oocytes (35.3% and 34.6%, respectively). The number of blastomer nuclei in embryos obtained from the PB+ group (52.0) was significantly higher than that of the PB− group (29.1); however, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ significantly (1:1.9 and 1:2.2, respectively) (P < 0.05). Chromosome analysis revealed that PB+ blastocysts had significantly more diploid blastomeres (69.7%) than PB− blastocysts (44.0%), whereas PB− blastocysts had significantly more triploid cells (34.0%) compared with PB+ oocytes (8.4%)(P < 0.05; χ2 test). These results indicate that porcine oocytes arrested at the M-I stage undergo cytoplasmic maturation during culture and have the same ability to develop to blastocysts after IVF as M-II oocytes but with a lower cell number; the latter might be caused by the slower embryonic development.

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MON-010 Effects of Delta-9-Tetrahydrocannabinol (THC) on Oocyte Competence and Early Embryonic Development

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.507 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Megan Misner ◽

Laura A Favetta

Keyword(s):

Embryonic Development ◽

Transcriptome Analysis ◽

Cannabis Use ◽

Reproductive Age ◽

Early Embryonic Development ◽

Mrna Levels ◽

Oocyte Competence ◽

Significant Difference ◽

The Impact

Abstract Cannabis is the highest used recreational drug amongst individuals of reproductive age. Fertility clinics advise against cannabis use when undergoing fertility treatments, but the literature backing this statement is weak. This rise in cannabis use has occurred simultaneously with the increase in the percentage of the main psychoactive component of cannabis, delta-9 tetrahydrocannabinol (THC) (1). Current literature suggests that THC mimics the effects of endogenous cannabinoids, binding to cannabinoid receptor 1 (CB1), which has been identified in reproductive tissues (2). Our research aims to study the impact of THC on oocyte maturation and pre-implantation embryonic development. An in vitro bovine system was used as it is the most appropriate translational model to humans for in vitro reproductive toxicity studies. Bovine oocytes were collected and matured under five treatment groups: control, vehicle (1:1:18 ethanol: TWEEN: saline), low THC (0.032uM), mid THC (0.32uM) and high THC (3.2uM). These doses mimic plasma concentrations reached after therapeutic (0.032uM) or low/high recreational (0.32uM and 3.2uM) cannabis use (3). We hypothesise that THC affects oocyte competence and proper early embryonic development in vitro. A negative THC dose-dependent response in cleavage rate was observed, with the highest THC group cleaving at 70.2% rate compared to 86.8% and 85.5% of control and vehicle groups, respectively (p<0.0001, n=7). There was no significant difference in blastocyst rate, suggesting that oocyte THC exposure affects the numbers of oocytes capable of development, but those able to cleave will properly reach blastocyst stage. We analyzed changes in gene expression, i) by a full RNA transcriptome analysis (24,128 transcripts screened) and ii) by quantification of Connexin 37 (CX37) and 43 (CX43) mRNA levels. Connexin expression is correlated to oocyte competence (4). RNA transcriptome analysis showed 62 genes that were significantly downregulated only in the low THC group. CX mRNA levels were measured via droplet digital PCR in both cumulus-oocyte complexes (COCs) and blastocysts. No significant differences were detected in blastocysts, however, a significant decrease in both CX37 and CX43 levels was measured in the low THC group in COCs (p<0.05, n= 9). Differences seen exclusively at the low THC dose suggest a role of THC as partial agonist of CB1. This research aims to understand the effects of cannabis on fertility, as current knowledge during pre-implantation development is limited, making it difficult for physicians to properly advise patients undergoing IVF. Reference: (1) ElSohly et al., Biol Psychiatry. 2016 Apr 1;79(7):613–9. (2) El-Talatini et al., PLoS ONE. 2009 Feb 24;4(2):e4579. (3) Whan et al., Fertil Steril. 2006 Mar;85(3):653–60. (4) Wang et al., Am J Physiol Endocrinol Metab. 2012 Jun 15;302(12):1511–8

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