Number Of Blastomeres Research Articles

Selection of viable in vitro-fertilized bovine embryos using time-lapse monitoring in microwell culture dishes.

Conventionally, in vitro-fertilized (IVF) bovine embryos for transfer are morphologically evaluated at day 7–8 of embryo culture. This method is, however, subjective and results in unreliable selection. We previously described a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse monitoring in our specially developed microwell culture dishes (LinKID micro25). The system can noninvasively identify prognostic factors that reflect viability after transfer. By assessing a combination of identified prognostic factors —timing of the first cleavage; number of blastomeres at the end of the first cleavage; and number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle— the pregnancy rate was improved over using conventional morphological evaluation. Time-lapse monitoring with LinKID micro25 could facilitate objective and reliable selection of healthy IVF bovine embryos. Here, we review the novel bovine embryo selection system that allows for prediction of viability after transfer.

114 GETTING THE YOLK OUT: THE USE OF A SOY LECITHIN-BASED CRYOMEDIUM FOR SEMEN BANKING IN THE PALLAS’ CAT AND FISHING CAT

Classically, semen from nondomestic felids has been frozen in an egg yolk-based cryomedium (TEY). However, the inclusion of egg yolk is problematic, resulting in possible batch to batch variation, microbial contamination, and regulatory concerns with international semen transport. Our previous research revealed that a soy lecithin-based cryopreservation medium (SOY) was superior to TEY for freezing domestic cat sperm. However, there is limited information on the efficacy of SOY in nondomestic felids. In the present study, our aim was to assess the effect of SOY v. TEY on motility, acrosome status, and fertilizing capacity of frozen-thawed sperm in 2 wild cat species: the Pallas’ cat (Otocolobus manul) and fishing cat (Prionailurus viverrinus). Semen was collected via electroejaculation from male cats (n = 3/species), split into 2 aliquots, extended in either SOY or TEY (containing 4% glycerol), slow-cooled, and frozen in straws over liquid nitrogen vapor. Sperm motility (percent progressively motile, PM; rate of progressive motility on 1 to 5 scale, RPM) was evaluated at 0, 1, 3, 6, and 24 h post-thaw and acrosome status (AS) was assessed via fluorescein isothiocyanate-peanut agglutinin staining at 0 and 6 h post-thaw. Heterologous IVF was performed using oocytes (n = 10–15/experimental unit, 2 replicates) collected laparoscopically from gonadotropin-treated domestic cats. At 48 h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm bound to the zona pellucidae of embryos and mature oocytes. The PM, RPM, and AS were analysed with repeated-measures ANCOVA, using pre-freeze values as a covariate. Embryo cleavage % and accessory sperm number were analysed with ANOVA. All data are reported as mean ± s.e. In the Pallas’ cat, PM, RPM, and AS of SOY-treated sperm (35.0 ± 7.6% motile, 2.8 ± 0.4 RPM, 41.2 ± 5.4% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (36.7 ± 4.4% motile, 2.8 ± 0.4 RPM, 45.3 ± 9.4% intact; 0 h) at any post-thaw time point. Similarly in the fishing cat, post-thaw PM, RPM, and AS of SOY-treated sperm (38.3 ± 7.3% motile, 2.6 ± 0.2 progression, 20.0 ± 2.9% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (31.7 ± 8.7% motile, 2.4 ± 0.3 RPM, 18.3 ± 6.4% intact; 0 h) at any time point. In the Pallas’ cat, neither embryo cleavage % (42.6 ± 7.0% SOY; 61.4 ± 14.8% TEY) nor accessory sperm number (12.2 ± 2.8 SOY; 18.1 ± 4.0 TEY) differed (P > 0.05) between treatments. Fishing cat results were similar, with no difference (P > 0.05) between SOY and TEY for cleavage % (60.4 ± 9.4% SOY; 47.9 ± 5.0% TEY) or accessory sperm number (4.6 ± 1.0 SOY; 5.5 ± 1.0 TEY). Collectively, these findings demonstrate that our SOY medium is an effective alternative to TEY for sperm cryopreservation in 2 nondomestic felid species. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free option represents a significant advance in quality control and biosecurity for cat semen banking and may enhance the use of assisted reproductive technologies for population management of imperiled felids. Research was funded by the Institute of Museum and Library Services.

150 PROTEOME OF BOVINE CUMULUS CELLS AS RELATED TO OOCYTE MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION

The present study was conducted to study the effect of cumulus-oocyte complex (COC) morphology on subsequent in vitro embryo development and to assess the proteome of their corresponding cumulus cells (CC). Cow ovaries were obtained at an abattoir and COC aspirated from 3–8 mm follicles. The COC were defined as type I (TI): homogeneous ooplasma and ≥4 layers of compact CC; type II (TII): granular ooplasma and ≥4 layers of slight expanded CC. Fifty COC had ~500,000 CC. Cumulus cells were frozen in ammonium bicarbonate and immediately lyophilized for proteome analysis. Other selected COC were matured in vitro in TCM-199-supplemented media for 24 h. After maturation, CC were collected (T24) and processed as described above. The remaining COC were fertilized with sperm from a fertile bull and zygotes, cultured in vitro until Day 7. Ten blastocysts per group were stained (Hoechst 33342) and blastomeres, counted for assessment of embryo quality. The CC proteins were obtained from the following groups: immature type I (TIT0) and type II (TIIT0), and in vitro matured type I (TIT24) and type II (TIIT24). For protein extraction, we used sonication (30 min, 4°C), freezing and unfreezing in liquid nitrogen, and maceration. The CC proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by ESI-MS/MS. Differences in cleavage, embryo rates, and blastomere numbers were analysed by t-test. Protein expression difference was set at 2.5-fold (P < 0.05). In silico protein interactions were investigated using STRING v. 10.0. There were no differences in cleavage (88 ± 4 v. 89 ± 8%) and embryo rates (36 ± 7 v. 33 ± 8%) between COC of TI (n = 220) and TII (n = 161), respectively. Blastomeres were also similar in TI (101) and TII (104) groups. Major proteins expressed in all CC were α-enolase, β-actin, oestradiol 17-β-dehydrogenase 1, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, heat shock protein β-1, histone H2B type 1-N, histone H4, mitochondrial malate dehydrogenase 2, protein disulfide isomarase A6, triosephosphate isomerase, tubulin α-1C chain, and vimentin. Glyceraldehyde 3-phosphate dehydrogenase appeared to be more expressed in TIT0, whereas tubulin α-1C chain and vimentin had greater expression in TIIT24. As evidenced by in silico analysis, most CC proteins interact among themselves, participating in complex networks involving intracellular signalling and other events. In conclusion, there are no difference in embryo development when using compact and early-expanded COC, indicating that both types can be selected for IVP. Protein profile of cumulus cell may serve as a marker for in vitro embryo competence.

154 COMPARISON OF DIFFERENT CULTURE MEDIA AND INCUBATION METHODS ON CULTURING MURINE EMBRYOS IN VITRO USING STRAW AS A RECEPTACLE

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P > 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

Influence of post-insemination nutrition on embryonic development in beef heifers

Previous studies have demonstrated that a decrease in nutrition immediately following AI reduces pregnancy success in beef heifers. The objective of this experiment was to determine if nutrient restriction following AI impacted early embryonic development among non-super ovulated heifers. Beef heifers in eight replications (Rep; Rep 1; n = 14, Rep 2; n = 15, Rep 3; n = 15, Rep 4; n = 14, Rep 5; n = 15, Rep 6; n = 15, Rep 7; n = 25, Rep 8; n = 25) across two locations (UMN, SDSU) were developed in a dry-lot and fed 125% NRC requirements from weaning to timed-AI (d 0). Heifers were timed-AI to a single sire in all replications. Immediately following AI, heifers were assigned, based on age, weight, and estrous response to one of two post-AI nutritional treatments. Half the heifers in each replication continued on the pre-insemination diet, serving as the control treatment (CON) and the remaining heifers were restricted to a sub-maintenance diet (RES). At UMN, heifers in the RES treatment were fed the same diet, but intake was limited to 80% NEm, while at SDSU, DMI remained the same, but diet composition was altered with the addition of straw to reduce NEm to 50% of requirements. On d 6, single embryos were collected nonsurgically and recovered embryos (CON; n = 46, RES; n = 42) were evaluated to determine quality (grade 1–9) and stage (1–4). Embryos were then stained and evaluated to determine the number of dead cells and total blastomeres. In Reps 1 through 6, concentrations of IGF-1 were assessed on d 0 and 6 and progesterone concentrations on d 4 and 6. Data were analyzed using the Mixed procedures of SAS. There were no treatment by Rep or treatment by location interactions for any embryo parameter evaluated, thus all data were pooled. Embryo stage and quality were improved (P < 0.01) in the CON (4.4 ± 0.16, 2.2 ± 0.19, respectively) compared to RES treatment (3.7 ± 0.16, 2.9 ± 0.19, respectively). Embryos in the CON treatment had greater total blastomeres (66.9 ± 5.05; P < 0.01) and tended to have a greater percentage of live cells (P < 0.10; 80.9 ± 4.19%) compared to RES (47.9 ± 5.41; 69.7 ± 4.39%, respectively). Progesterone and IGF-1 concentrations did not differ between treatments. In summary, nutrient restriction for 6 days immediately following AI resulted in poorer quality embryos that were delayed in stage of development, suggesting that immediate changes in nutritional status after insemination can alter early embryonic development.

Embryo multinucleation at the two-cell stage is an independent predictor of intracytoplasmic sperm injection outcomes

To determine the prognostic impact of the nuclear status at the two-cell stage on intracytoplasmic sperm injection (ICSI) outcomes. Retrospective study. Hospital. Only ICSI cycles with time-lapse monitoring of transferred embryos with known implantation/delivery data from November 2012 to December 2014 were included. A total of 2,449 embryos were assessed for multinucleation rates at the two- and four-cell stage, and 608 transferred embryos were studied for ICSI outcomes. None. Implantation rate (IR) and live birth rate (LBR) according to the number of multinucleated blastomeres at the two-cell stage: none (Without-MNB2cell), one (MNB1/2cell), and two (MNB2/2cell); morphokinetics of MNB2cell embryos. Embryos with MNB1/2cell led to lower IR (27.7%) and LBR (22.7%) than embryos Without-MNB2cell (33.4% and 29.8%, respectively). The MNB2/2cell embryos led to significantly lower IR (18.3%) and LBR (13.4%) than embryos Without-MNB2cell. This difference remained significant in multivariate analysis for implantation (odds ratio 0.57; 95% confidence interval 0.34-0.94) and birth (odds ratio 0.46; 95% confidence interval 0.26-0.80), independently of the other significant parameters (women's age, time of two-cell formation, and multinucleation at the four-cell stage). Among implanted MNB2cell, if cleavage into four cells occurred later than 37hours after insemination, embryos were significantly more likely to lead to birth. The presence of multinucleation at the two-cell stage and more specifically in both blastomeres had a significant negative impact on birth potential. Thus, embryo multinucleation at the two-cell stage should be used as an additional noninvasive criterion for embryo selection.

Sex differences in response of the bovine embryo to colony-stimulating factor 2.

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine.

T-2 mycotoxin slows down the development of mouse blastocysts, decreases their blastomere number and increases chromatin damage.

The mycotoxin T-2 has many harmful effects on mammalian cells and reproductive functions. In the present study, the in vitro effect of T-2 toxin on mouse blastocysts was examined. Embryos were cultured in media supplemented with 0.5, 0.75 and 1 ng/ml T-2. Different exposure times were applied [96 h (treatment I) or 24 h following 72 h in toxin-free media (treatment II)]. Blastomere number, nuclear chromatin status and blastocoel formation were investigated in blastocysts. Our data show that the effect of T-2 toxin may vary depending on the stage of the embryo at the start of exposure. At 96 h of exposure, the blastocysts had blastomeres with normal chromatin quality but their developmental potential was decreased. After 24 h of exposure applied following a 72-h culture, blastomeres had a higher level of chromatin damage, although their developmental potential was the same as in the control embryos. In both cases, decreased mitotic rate was found, which resulted in decreased blastomere number even at low toxin concentration.

Fragment removal of the fragmented human day2 embryos significantly increased subsequent development and clinical outcomes

The purpose of this study was to assess the beneficial effect of fragment removal on the subsequent development and pregnancy outcome of the fragmented human day 2 embryos. The study included 96 patients who underwent day 3 ET program from March 2015 to October 2015. The patients were divided into two groups; Fragment removal (FR) group (n=48) and control group (n=48). There were no differences between the FR and the control groups in the number of blastomeres (4.0±1.3 vs. 4.0±1.3) and the morphological grade of embryos (3.1±0.6 vs. 3.1±0.5) before fragment removal on day 2. Fragment removal was performed on the grade 3 embryos (10 %< degree of fragmentation <25%) of the FR group. We performed assisted hatching with laser and fragments removal on day 2. Before fragment removal, the fragmented embryos were incubated in G-PGD medium (Vitrolife) under paraffin oil (Ovoil, Vitrolife) for 30 minutes. Microsurgical fragment removal was performed with handmade suction micro-pipet with outer diameter of 30μm. There were no differences in the characteristics of the patients between the FR and the control groups; mean age of the patients (36.2±4.3 vs. 36.2±4.1), endometrial thickness (10.4±2.5 vs. 10.0±2.0), AMH levels (4.4±4.5 vs. 4.2±2.9), and mean number of retrieved oocytes (8.7±5.7 vs. 9.2±4.8). No differences were also observed between the two groups in fertilization rate (76.0±17 vs. 71.0±22%) and mean number of ET (2.0±0.4 vs. 2.0±0.3). After fragment removal and 24 h culture in vitro, although the number of blastomeres (7.0±1.6 vs. 6.8±1.6) was comparable in the two groups, embryo morphological grade (2.0±0.8) of the FR group was significantly improved compared to that (3.2±0.5 p<0.01) of the control group. Clinical pregnancy (43.8%) and implantation rates (28.1%) of the FR group were significantly higher than the rates (23.0 and 14.7%, respectively, P<0.05) of the control group. Early fragment removal on day 2 significantly improved the subsequent development and pregnancy outcomes of the fragmented embryos. These results suggest that early fragment removal effectively prevents secondary degeneration of fragmented embryos by eliminating the release of toxic substances as soon as possible.

Sequential embryo scoring system can substitute for time lapse monitoring system

Time lapse scoring system has an advantage in selecting the best embryo by evaluating embryo's continuous development at certain time points. In our laboratory, we investigated if the sequential embryo-scoring system could exhibit positive effects in selecting embryos by replacing the time-lapse function with cleavage stage embryos, including early-cleavage at certain time points. All fresh, non-donor IVF cycles performed from January 2015 through August 2015 (n=485). After insemination, embryos were scored at the following 3 time points: 2-cell cleavage (26h), day 2 (40-41h), and day 3 (64-65h). The highest score of the embryo at 3 time points is as follows: 2-cell cleavage (2 points) + day 2 (5 points) + day 3 (5 points) = 12 points. In two embryos transfer, the highest total score is 24 points. The score was dependent on three factors: (number of blastomere, synchrony of cleavage, level of fragmentation). Scoring was done by subtracting scores from embryos with one or more defects. To determine a cut-off value, pregnancy rate was evaluated in one point unit from 6 points to 24 points based on two embryos transfer. Based on the cut-off value, embryos were divided into two groups, and the pregnancy and implantation rate were checked. The cut-off value was 16 points, and it was confirmed that pregnancy rate significantly improved. When the two groups (group (1) scored higher than 16 points, group (2) scored less than 15 points) based on the cut-off value were divided and compared. Patient characteristics were similar in the 2 groups. In each group, age (Group 1: 35.6±2.0 vs. Group 2: 36.0±1.9) basal AMH conc. (4.2±2.9 vs. 4.8±2.8ng/ml), and endometrial thickness (10.2±2.2 vs. 10.1±2.0mm) did not show significant differences. There were no significant differences between 2 groups in the number of MII oocytes (10.6±6.0 vs. 10.6±5.3) and number of embryo transfer (2.0±0.1 vs. 2.0±0.1). Group (1) had a significantly high pregnancy rate (68.8%, P=0.001) and implantation rate (57.1%, P=0.001) compared to that of Group (2) (41.6% and 31.6%, respectively). Our study evaluated the continuous development process of the embryo through the sequential embryo scoring system. We found that embryos with scores higher than 16 points resulted in a significantly high pregnancy rate. Therefore, the sequential embryo scoring system is effective in predicting high pregnancy rates.

Live birth rate and number of blastomeres on day 2 transfer.

To investigate whether the presence of large fragment (LF) and abnormal cell divisions (ACDs) has influenced the correlation between live birth rate and number of blastomeres detected on day 2 by conventional scoring. This study included 578 embryos cultured in time lapse and selected for transfer by conventional scoring on day 2. By time-lapse recordings, embryos were reassessed to identify ACDs and/or LFs mistaken as blastomeres. The latter identifications were used to recalculate fragmentation rate and the number of blastomeres. Life birth rate according to number of blastomeres was compared in (a) embryos selected by conventional scoring and (b) embryos reassessed by time lapse. After conventional scoring, embryos with four cells had a significantly higher pregnancy rate than embryos with less than four cells and embryos with more than four cells. By time-lapse assessment, ACDs and/or recalculated fragmentation >25% was recognized in 106/578 (18.3%) of transferred embryos. None of them resulted in a live birth. After exclusion of these embryos, the number of blastomeres on the day of transfer did not have any impact on life birth rate. Conventional scoring on day 2 did not detect ACDs and LFs mistaken as blastomeres. LFs can lead to a recalculated fragmentation rate to >25%. No significant correlation between live birth rate and number of blastomeres in day 2 embryos was observed when embryos with ACDs and fragmentation >25% were excluded. Recognition of ACDs and fragmentation >25% is more predictive of live birth than number of blastomeres.

Requirement of Leukemia Inhibitory Factor or Epidermal Growth Factor for Pre-Implantation Embryogenesis via JAK/STAT3 Signaling Pathways.

Leukemia inhibitory factor (LIF) plays a key role in the survivability of mouse embryos during pre-implantation. In this study, we verified the role of LIF by detecting gene expression in morula stage embryos through DNA microarray. Our results showed that LIF knockdown affected expression of 369 genes. After LIF supplementation, the epidermal growth factor (EGF) is most affected by LIF expression. To observe the correlation between LIF and EGF, the LIF knockdown embryos were supplemented with various growth factors, including LIF, EGF, GM-CSF, TGF, and IGF II. Only LIF and EGF caused the rate of blastocyst development to recover significantly from 52% of control to 83% and 93%, respectively. All of the variables, including the diameter of blastocysts, the number of blastomeres, and cells in ICM and TE, were almost restored. Moreover, EGF knockdown also impaired blastocyst development, which was reversed by LIF or EGF supplementation. The treatment with various signaling suppressors revealed that both EGF and LIF promoted embryonic development through the JAK/STAT3 signaling pathway. These data suggest that the EGF and LIF can be compensatory to each other during early embryonic development, and at least one of them is necessary for sustaining the normal development of pre-implantation embryos.

Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts.

The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre-implantation feline embryos obtained from 6th to 9th day of invitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

SummaryPrimordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model.

Nop2 is required for mammalian preimplantation development.

Nucleolar protein 2 (NOP2) is evolutionarily conserved from yeast to human, and has been found to play an important role in accelerating cell proliferation, cell-cycle progression, and tumor aggressiveness. The expression pattern and function of Nop2 during early mammalian embryo development, however, has not been investigated. We identified Nop2 as an essential gene for development to the blastocyst stage while performing an RNA interference (RNAi)-based screen in mouse preimplantation embryos. Nop2 is expressed throughout preimplantation development, with highest mRNA and protein accumulation at the 8-cell and morula stages, respectively. RNAi-mediated knockdown of Nop2 results in embryos that arrest as morula. NOP2-deficient embryos exhibit reduced blastomere numbers, greatly increased apoptosis, and impaired cell-lineage specification. Furthermore, knockdown of Nop2 results in global reduction of all RNA species, including rRNA, small nuclear RNA, small nucleolar RNA, and mRNA. Taken together, our results demonstrate that Nop2 is an essential gene for blastocyst formation, and is required for RNA processing and/or stability in vivo during preimplantation embryo development in the mouse.

96 EFFICIENT PRODUCTION OF MONOZYGOTIC TWIN BOVINE EMBRYOS USING BLASTOMERE SEPARATION TECHNIQUE WITH COMMERCIAL WELL OF THE WELL CULTURE DISH

Monozygotic twin embryos can be produced using the technique of blastomere separation and well of the well (WOW) dish having handmade micro-wells by the needle depression (Tagawa et al. 2008). We have recently reported that developed commercial WOW dish enhances embryo competence in individual culture (Sugimura et al. 2010). The present study was conducted to evaluate the availability of commercial WOW dish for production of monozygotic twin embryos in bovine. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and IVC. For each culture, TCM-199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10 mg mL–1 BSA, and CR1aa containing 5% CS were used. To evaluate the adaptability of dishes on culture of isolated blastomeres from different cell stage, 2- (n = 63), 4- (n = 94), 8- (n = 137), and 10- to 14- (n = 116) cell stages were obtained on 24–27 h, 30–36 h, 48–54 h, and 48–54 h from the beginning of fertilization, respectively. The zona pellucida was removed by exposure of 0.25% pronase, followed by gentle pipetting by inspiration and expiration in the IVC medium. Then, two halves separated from the original number of blastomeres were randomly allocated to the conical micro-wells of commercial dish (Dai Nippon Printing, Tokyo, Japan) or created micro-wells by pressing the bottom of the dish with an eyeleteer (control). The approximate diameter and depth of each 25 wells in a commercial dish was 287 and 168 μm, and each 20 wells in the control were 800 and 600 μm. The blastomeres were cultured in wells covered with a droplet of 2.5 μL well–1 IVC medium until Day 8 (IVF = Day 0). Expanded blastocysts (n = 28) derived from tetra-blastomeres of 8-cell stage were stained to determine the number of the inner cell mass (ICM) and trophectoderm (TE) in each group. Statistical significance of the blastocyst formation rates and the number of cells were analysed by the chi-square test and the Student’s t-test, respectively. In the 2-cell stage, blastocyst formation rate in commercial dish tended to be higher than that in the control (60.0% v. 46.1%). The rate of monozygotic blastocyst pairs in commercial dish was higher compared with the control (48.0% v. 26.3%, P &lt; 0.05). In the 4-cell stage, rates of blastocyst formation (50.0% v. 33.0%, P &lt; 0.05) and the pairs (39.5% v. 12.5%, P &lt; 0.01) in the commercial dish, both were higher compared with the control. In the 8-cell stage, there were no differences between two groups in rates of blastocyst formation (53.1% v. 59.0%) and the pairs (41.8% v. 48.7%), similarly in the 10- to 14-cell stage (47.9% v. 56.8% and 36.2% v. 40.9%, respectively). The ICM, TE, and total cell numbers were not different between the commercial and the control dish (28.0 ± 3.2 v. 26.0 ± 3.8, 64.6 ± 4.3 v. 76.0 ± 7.9, and 92.6 ± 6.2 v. 102.0 ± 11.0, respectively). These results indicate that separated blastomeres could be developed to blastocysts efficiently and stably regardless of embryo cell stage with a commercial WOW culture dish.

104 EMBRYO MANIPULATION TECHNIQUES ALTER PRE-IMPLANTATION DEVELOPMENT AND GENE EXPRESSION IN MOUSE EMBRYOS

Micromanipulation techniques are commonly employed with human embryos; however, the long-term effects on growth and development of embryos are unknown. Subjective techniques, such as embryo grading, may not be sensitive enough to show differences in developmental potential and more objective assessments are needed. The objective of this experiment was to evaluate whether manipulation techniques and cryopreservation alter morphologic and gene expression endpoints of embryos. Two-cell mouse embryos were cultured to blastocyst stage and divided into 6 treatment groups: nonmanipulated control (UNMAN; n = 252), laser-assisted hatching (LAH; n = 124), LAH with slow cooling cryopreservation (LAHcryo; n = 78), simulated biopsy (BIOP; n = 90) performed by laser disruption of 1/8 of the embryo’s cellular volume, BIOP with slow cooling cryopreservation (BIOPcryo; n = 91), and BIOP with vitrification (BIOPvit; n = 66). After manipulation or thaw, embryos were cultured 28 h to blastocyst stage. Fully hatched and hatching blastocysts were collected for cell counts and quantitative PCR to assay absolute transcript abundance for Plac8, Glut1, Oct4, and Cox2, 18s rRNA, and Ppia using standard curves generated from serial dilutions of digested plasmids. Data were analysed using ANOVA with Duncan’s post hoc test and significance was defined as P &lt; 0.05. Manipulation groups differed in developmental stage at 28 h postmanipulation (P = 0.03), with a larger percentage of embryos completely hatched in LAH (46%) and BIOP (45%) groups compared to UNMAN (11%). Cryopreservation reduced percentages of completely hatched embryos (LAHcryo = 26%; BIOPcryo = 31%; BIOPvit = 19%). The number of cells per embryo also varied (P &lt; 0.001); UNMAN and LAH groups had similar numbers of blastomeres (UNMAN = 71; LAH = 74), whereas BIOP groups had reduced cell counts (BIOP = 53; BIOPcryo = 56; BIOPvit = 48). Patterns of gene expression varied between groups; the BIOP group had more cDNA per cell (UNMAN = 125 ng cell–1; BIOP = 202 ng cell–1; P = 0.004), whereas BIOPcryo group did not show this effect (BIOPcryo = 111 ng cell–1). Transcript abundance of Cox2, Oct4, and Glut1 per embryo was significantly decreased (P &lt; 0.05) in biopsied embryos that were cryopreserved, either by slow-cooling or vitrification (Cox2: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268; Oct4: UNMAN = 266, BIOP = 181, BIOPcryo = 95, BIOPvit = 97; Glut1: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268). The data suggests that micromanipulation alters developmental timelines and gene expression of embryos. While manipulations differ in degree and nature of effect, our investigation implies all manipulations have some effect on the transcriptome of the developing embryo. Differences in cDNA quantity in the BIOP group may indicate a stress response or recovery attempt that becomes blunted in biopsied embryos which are subsequently cryopreserved. Thus, our study suggests that cryopreservation of biopsied embryos may come at a price and offers scientific support for only judiciously selected and medically indicated embryo biopsies.

Morphokinetics of cloned mouse embryos treated with epigenetic drugs and blastocyst prediction.

Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success.

Morphokinetics analysis of embryos derived from vitrified/warmed oocytes.

Oocyte vitrification is a worldwide used technique that has proved its worth. Although it was shown not to alter oocyte integrity, a recent study concluded that it may affect oocyte embryo development. As the morphology and kinetics of embryos derived from sibling fresh and vitrified oocytes have not been described previously, the present study evaluates cleavage rate, blastomeres size, fragmentation rate, and blastocyst formation in vitrified/warmed oocyte derived embryos (VODE) as compared with sibling fresh oocytes derived embryos (FODE). This investigation included 90 infertility cases displaying large cohort of mature oocytes at pick up, divided into 2 groups after denudation. A part of oocytes underwent ICSI while others were vitrified. Oocyte warming cycles were performed when no pregnancy was achieved using fresh eggs. Zygote to blastocyst development was recorded prospectively in an image database up to day 5. VODE did not show major difference as compared with FODE in terms of cleavage rate, number of blastomeres, fragmentation rate, and blastomeres size. Furthermore, percentage of morulae at day 4 and blastocysts at day 5 are not affected by oocyte vitrification. Although our results show that embryo development is not altered by oocyte vitrification, offspring follow-up is essential to exclude any adverse developmental effect of the technique.

The effects of paternal obesity on sperm characteristics and early embryonic development: results from a retrospective study

In humans, it is widely demonstrated that maternal obesity negatively affects fertility, pregnancy outcomes and offspring's health. Less is known about the effects of paternal obesity on fertility and offspring's development. Recent observations in animal models show that diet-induced obese males have impaired sperm quality, delayed preimplantation growth of the fertilized oocyte, and reduced implantation rate. Our aim was to evaluate the effects of paternal obesity on sperm quality and subsequently embryonic development and implantation in humans. Body mass index (BMI), sperm and embryo parameters of 192 men who participated in a fertility program (IVF or ICSI) at the Leuven University Fertility centre between March and September 2014 were evaluated. Sperm parameters included: concentration, total sperm count, motility and morphology. Embryo parameters included number and asymmetry of blastomeres, degree of fragmentation and implantation success. The embryonic development parameters were obtained using a new computer assisted scoring system (CASS) based on multilevel image analyses. Our analytic study included sperm data from 192 men and embryo characteristics from 138 couples. Multiple regression models were used to determine potential associations between paternal obesity/BMI and sperm or embryo parameters. No significant associations were found between paternal obesity/BMI and the sperm parameters measured. After adjusting for potential confounding, we found a borderline significant effect of paternal obesity/BMI on the degree of asymmetry of blastomeres: coefficient was +0.05 (p=0.047) if men were obese, and coefficient was +0.004 (p=0.059) with increasing paternal BMI. This effect was only seen on day 2 of embryonic development. No effect of paternal obesity/BMI on implantation success was found. In order to confirm a potential effect of obesity on embryonic development our analyses will be further extended on a larger dataset. We further aim to gain insights on the underlying epigenetic mechanisms that have been reported as being mediated by paternal obesity.