Ratio Of Inner Cell Mass Research Articles

#181 : The Importance of Inner Cell Mass Ratio for Embryo Grade Prediction Using Artificial Intelligence

Background and Aims: While reports suggest that the Inner Cell Mass (ICM) ratio may impact pregnancy rates, the Gardner criteria for grading ICM does not specify the ICM size or ratio. This study investigates the potential impact of the ICM ratio on predicting ICM grades using artificial intelligence. Methods: The study analyzed 1,963 Day 5 blastocyst images obtained from seven IVF clinics between June 2011 and May 2022. The images were matched with metadata, including ICM grade and ICM ratio. ICM ratio was computed as the ratio of the ICM area to the embryo area. ICM was graded according to the Gardner criteria and only ICM A and C grade groups were used to avoid ambiguity. Two CNN (Convolutional Neural Network) models were built: one with embryo images including ICM ratio and one without. Results: The use of ICM ratio in conjunction with the original grading model resulted in an improved model performance. Logistic regression analysis revealed the ICM ratio as a significant variable impacting embryo grade, with a p-value of less than 0.001. The coefficient for ICM ratio demonstrated an 8-fold increase in the odds ratio for grade A embryos with every 1-unit increase. Conclusions: We demonstrated that the CNN model could predict ICM grades with fair accuracy and that the inclusion of the ICM ratio significantly improved its performance. While CNNs are widely used for image analysis by extracting features from patterns, they may not effectively learn the characteristics of an area. To capture the important area-related features, measured values should be considered along with the image. Furthermore, the ICM ratio appears to be a significant feature that embryologists consider when grading embryos in clinical settings, beyond the Gardner criteria. Additional studies are warranted to investigate other features for evaluating ICM.

ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows.

Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.

MITOQUINONE RESCUES STRESS INDUCED MITOPHAGY IN AGED OOCYTES

It is well established that decreased oocyte quality from advanced maternal age (AMA) women are associated with dysfunctional mitochondria. Mitophagy is a critical pathway to regulate mitochondrial quality. Our previous research demonstrated reduction in key mitophagy protein Parkin (PRKN) and its activity in oocytes from AMA women. The objective of this study was to investigate if maturing aged mouse oocytes with Mitoquinone (MQ) would improve oocyte mitochondrial quality by rescuing mitophagy. First, oocytes from young (2 months) and aged (13.5 months) mice were matured with or without MQ and the levels of p-PRKN and PRKN were analyzed by Western blot (Jess, ProteinSimple). In the second experiment, young oocytes with or without MQ were matured under normal (6.5%) or high oxygen (20%) environment, and the expression of p-PRKN and PRKN, mitochondria DNA (mtDNA) copy number (analyzed by qPCR) and blastocyst development after in vitro fertilization, blastocyst hatching, and inner cell mass (ICM) ratio were examined. We observed a significant decrease in p-PRKN (0.32±0.09, p≤0.01) and PRKN (0.30±0.1, p≤0.05) in aged compared to young mouse oocytes (normalized to 1). Treatment of aged oocytes with MQ rescued p-PRKN (1.11±0.19 p≤0.01) and PRKN (0.67±0.04, p≤0.05) protein expression. Oocytes matured under high oxygen environment exhibited significantly reduced p-PRKN (0.5±0.1, p≤0.05) compared to oocytes matured under normal oxygen conditions. This coincided with an increase in mtDNA copy number (Young: 56957, High oxygen: 72758 copies). MQ treatment significantly increased p-PRKN protein expression (1.2±0.2, p≤0.05) as well as decreased mtDNA copy number (50312 copies) in oxidatively stressed oocytes. Addition of MQ significantly improved blastocyst development (High O2: 39.5%, High O2+MQ: 70%, p≤0.05), hatching (High O2: 59%, High O2+MQ: 93%, p≤0.05), and ICM ratio (High O2: 14.61%, High O2+MQ: 22.5%, p≤0.0001) of oxidatively stressed oocytes that showed decreased trends in blastocyst development potential. We have demonstrated previously that oocytes from AMA women (>38 years old) exhibited significantly less p-PRKN and PRKN than oocytes from young women (<32 years old). Similar PRKN expression pattern was obtained in young and aged mice and we also observed decreased p-PRKN in oxidatively stressed mouse oocytes, indicating that mitophagy via Parkin pathway is altered in aged and oxidatively stressed oocytes. We have shown for the first time that treating aged and oxidatively stressed oocytes with MQ rescues p-PRKN and PRKN protein expression to ameliorate mitophagy. MQ also improved blastocyst development potential of oxidatively stressed oocytes.

62 Investigating differences in gene expression between invitro-produced bovine embryos and parthenotes

Parthenogenic activation allows for the development of an oocyte without male gamete contribution and may serve as a suitable model for understanding maternal and paternal contributions during development. In the bovine, parthenotes lack the ability to survive to term once transferred into females. The goal of this study was to investigate the gene expression profile at the blastocyst stage in parthenotes and embryos by characterising expression of developmentally important genes, such as markers for pluripotency (OCT4, NANOG), hypoblast (GATA6), epiblast (SOX2), trophectoderm (CDX2), and maternal-embryo communication (IFNT2). To test this, IVM oocytes were either fertilized to a bull with known fertility invitro or activated. To activate, oocytes were denuded, and placed in ionomycin calcium salt for 5min. They were incubated for 3h in 6-(dimethylamino)purine (6-DMAP) and placed in synthetic oviductal fluid (SOF). Putative zygotes and activated oocytes were cultured in SOF for 8 days. Cleavage (at least one cellular division) was recorded on Day 3 and development to the blastocyst stage was recorded on Day 8 after insemination or activation. Cleavage rate was 85.9% and 83.2% for parthenotes and embryos, respectively. Both groups produced a similar blastocyst rate, 32.3% for parthenotes and 33.7% for embryos. On Day 8, blastocyst stage parthenotes and embryos were collected. Data were analysed by ANOVA (Tukey HSD post hoc test) using the GLM procedure of SAS version 9.4. Pools of 5 embryos or parthenotes (3 replicates) were flash frozen and stored until RNA isolation (PicoPure™ RNA Isolation Kit). Real-time PCR was used for quantification of gene expression. Genes were analysed relative to a housekeeping gene, GAPDH. There was no difference in gene expression detected for OCT4 (P=0.25), NANOG (P=0.11), GATA6 (P=0.32), SOX2 (P=0.25), or IFNT2 (P=0.52). Expression of CDX2 was lower in the parthenotes than the embryos (P=0.05). In a second experiment, the proteins GATA6, NANOG, and CDX2 were immunolocalized in 17 parthenotes and 15 embryos. Fixed embryos were permeabilized, blocked, and placed in primary antibodies overnight. After, they were placed into the secondary antibody for 1h, followed by nuclear stain. There was a decreased mean intensity of CDX2 in the parthenotes compared to embryos (P=0.005). No difference (P&amp;gt;0.05) in GATA6 or NANOG was observed between the 2 groups. The ratio of inner cell mass to trophectoderm was higher (P=0.04) in the parthenotes (2.5±0.23) than in the embryos (1.7±0.25). Here we analysed and confirmed the expression of developmentally important genes at the blastocyst stage in embryos and parthenotes. CDX2, a marker of the trophectoderm that will later give rise to the placenta, was downregulated in parthenotes. This highlights the importance of the contribution of the paternal genome to development. Further research is necessary to elucidate the ability of the parthenotes to establish and maintain pregnancy. This research was supported by the National Needs Fellowship funded by USDA NIFA Grant 2019-38420-28972.

Embryo selection in developing countries- inner cell mass to blastocyst dimension ratio as a predictor of pregnancy outcome

Is there a correlation between inner cell mass (ICM) to blastocyst dimensions (diameter and area) ratio and pregnancy outcome, which can be used to select embryo for transfer in developing countries? A prospective observational study was performed for a duration of 18 months at a tertiary level IVF centre. 185 subjects who had autologous or donor egg derived blastocyst transfers were included in the study. 385 Blastocysts were photographed at 116 hours post ICSI. ICM to blastocyst dimensions ratio was calculated using Image J software. Morphological scoring of embryos was performed according to Istanbul consensus. Predictive value of ICM to blastocyst diameter and area ratios to implantation and clinical pregnancy rate were studied by ROC curve analysis. Correlation coefficients were drawn between dimensions ratio and morphological scoring. Mean diameter ratio of ICM to blastocyst among embryos that gave clinical pregnancy was 0.33 +/- 0.05 as against 0.30 +/- 0.09 among embryos that did not give clinical pregnancy (p value – 0.015). Mean area ratio of ICM to blastocyst among embryos that gave clinical pregnancy was 0.14 +/- 0.04 as against 0.13 +/- 0.07 (p value – 0.26). Morphological scoring was calculated from grading as per Istanbul consensus and quantified as 1/ICM score x 1/ Trophectoderm score x Expansion score. Mean morphological scoring of embryos that gave clinical pregnancy was 2.45 +/- 1.27 as against 2.04 +/- 1.08 among embryos that did not give clinical pregnancy (p value – 0.03). Correlation coefficient between morphological scoring and mean diameter ratio of ICM to blastocyst was 0.23 (p - 0.003), showing mean diameter ratio is positively correlated with morphological scoring. Correlation coefficient between mean area ratio of ICM to blastocyst to morphological scoring was 0.14 (p - 0.06). Area under curve (AUC) for ICM to blastocyst diameter ratio to predict clinical pregnancy was 0.586 (p-0.04), for area ratio was 0.576 (p-0.04) and for morphological scoring was 0.595 (p-0.04). The predictive ability for clinical pregnancy was highest for existing morphological scoring followed by ICM to blastocyst diameter ratio and least for area ratio. Ratio between ICM to blastocyst dimensions would serve as a simple, cost-effective tool for selecting embryos prior to transfer in developing countries.

The histone lysine demethylase KDM7A is required for normal development and first cell lineage specification in porcine embryos

ABSTRACTThere is growing evidence that histone lysine demethylases (KDMs) play critical roles in the regulation of embryo development. This study investigated if KDM7A, a lysine demethylase known to act on mono-(me1) and di-(me2) methylation of H3K9 and H3K27, participates in the regulation of early embryo development. Knockdown of KDM7A mRNA reduced blastocyst formation by 69.2% in in vitro fertilized (IVF), 48.4% in parthenogenetically activated (PA), and 48.1% in somatic cell nuclear transfer (SCNT) embryos compared to controls. Global immunofluorescence (IF) signal in KDM7A knockdown compared to control embryos was increased for H3K27me1 on D7, for H3K27me2 on D3 and D5, for H3K9me1 on D5 and D7, and for H3K9me2 on D5 embryos, but decreased for H3K9me1, me2 and me3 on D3. Moreover, KDM7A knockdown altered mRNA expression, including the downregulation of KDM3C on D3, NANOG on D5 and D7, and OCT4 on D7 embryos, and the upregulation of CDX2, KDM4B and KDM6B on D5 embryos. On D3 and D5 embryos, total cell number and mRNA expression of embryo genome activation (EGA) markers (EIF1AX and PPP1R15B) were not affected by KDM7A knockdown. However, the ratio of inner cell mass (ICM)/total number of cells in D7 blastocysts was reduced by 45.5% in KDM7A knockdown compared to control embryos. These findings support a critical role for KDM7A in the regulation of early development and cell lineage specification in porcine embryos, which is likely mediated through the modulation of H3K9me1/me2 and H3K27me1/me2 levels, and changes in the expression of other KDMs and pluripotency genes.

Treatment of Buffalo (Bubalus bubalis) Somatic Cell Nuclear Transfer Embryos with MicroRNA-29b Mimic Improves Their Quality, Reduces DNA Methylation, and Changes Gene Expression Without Affecting Their Developmental Competence.

microRNA-29b (miR-29b) plays an important role in controlling DNA methylation in cells. We investigated its role during early embryonic development in buffalo embryos produced by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). miR-29b expression was highest at the 2-cell stage, decreased (p < 0.001) at the 4-cell stage, and remained low thereafter at the 8-cell, morula, and blastocyst stages, showing a similar pattern in cloned and IVF embryos. Treatment of reconstructed embryos with miR-29b mimic for 1 hour after 1 hour of electrofusion increased (p < 0.05) the total cell number and decreased (p < 0.05) the levels of apoptosis and DNA methylation compared with controls. It also increased (p < 0.05) the ratio of inner cell mass:trophectoderm cell numbers of blastocysts compared with controls to the levels observed in IVF blastocysts. However, the blastocyst rate was not affected by treatment with miR-29b mimic (29.0% ± 2.0% vs. 27.0% ± 2.0% for controls). The treatment decreased (p < 0.001) the expression of epigenetic-related genes, DNMT3A and DNMT3B, but not DNMT1, and increased (p < 0.05) that of pluripotency- (NANOG, OCT4, and SOX2) and development-related genes (FGF4 and GLUT1) in blastocysts compared with controls. Our results suggest that miR-29b mimic treatment of reconstructed embryos improves the quality, reduces the level of apoptosis and DNA methylation, and changes gene expression in SCNT blastocysts without affecting the blastocyst rate.

124 Cell Count of Bovine In Vitro-Produced Blastocysts After In Vitro Maturation in Glass versus Plastic Vials

Optimal bovine in vitro oocyte maturation (IVM) is a prerequisite for subsequent optimal blastocyst rates. Ovum pick-up (OPU), by which cumulus–oocyte complexes (COC) are collected in vivo, is performed outside a laboratory and often requires IVM to take place during transportation from the farm to the IVF laboratory. Hashem et al. (2017 Reprod. Fertil. Dev. 29, 179) demonstrated that blastocyst rates are affected by type of vial (glass v. plastic), number of COC per vial, and volume of medium per vial. This was achieved by maturing more than 2500 COC from slaughterhouse material under contrasting conditions, followed by standardised IVF and in vitro culture (IVC) and observation of blastocyst rates, morphology (1: poor; 2: good; 3: excellent), and kinetics (1: blastocyst; 2: expanded blastocyst ; 3: hatching/hatched blastocyst). Here we examined differential staining of a subset of expanded blastocysts (XB) from the previous study to assess the influence of vial material, medium volume, and number of COC per vial on total cell count, number and ratio of inner cell mass (ICM), and trophectoderm (TE) cells. In experiment 1 (4 groups), oocytes were matured in different vials without lids in an incubator at 5.5% CO2 in humidified atmospheric air at 38.5°C to assess plastic toxicity. In experiment 2 (6 groups) and experiment 3 (6 groups), the 2 best performing vials-polypropylene cryovials (Sigma-Aldrich, St. Louis, MO, USA) and glass vials (VWR International, Radnor, PA, USA)-containing 50% (Exp. 2) or 95% (Exp. 3) medium volume per vial and 5, 20, or 45 COC per vial were tested. In experiments 2 and 3, the vials were closed and incubated in atmospheric air at 38.5°C. All groups were evaluated for blastocyst rates, kinetics, and morphology. Because kinetics (range 2.01–2.25) and morphology (range 2.15–2.50) were similar in all groups, only XB were collected from each group. These were fixed and stained with CDX2 antibody and Hoechst (Wydooghe et al. 2011 Anal. Biochem. 416, 228-230) and their ICM and TE cells were counted. The cells were counted manually in blinded groups using an inverted fluorescence microscope and 16× magnification. Counts of total, ICM, and TE cells were compared between treatments by a two-way ANOVA analysis. A total of 240 XB from the 16 different vial groups were counted in the 3 experiments, with average total cell counts of 139 (110–211) and ICM cell counts of 44 (28–75). Even though the blastocyst rates differed between some of the groups, the cell counts within the XB did not differ statistically significantly between groups. In fact, the highest cell count was found in the glass vial group with the lowest blastocyst rate (45 COC per vial in 50% medium volume; blastocyst rate 28%, total cells 211, ICM cells 75). We have previously demonstrated that the type of vial, number of COC per vial, and the volume of medium per vial influence the subsequent blastocyst rates. It is concluded, however, that the embryos able to proceed to the blastocyst stages seem to be of the same quality in all groups, assessed by kinetics, morphology, and cell counts within XB.

Delayed blastocyst formation or an extra day culture increases apoptosis in pig blastocysts

In the present study, the timing was examined of blastocyst collection/formation or of how the duration of post-blastulation culture affected the quality and developmental competence of in vitro-produced pig parthenogenetic embryos. The earliest apoptotic signals were observed at the morula stage while the earliest cytoplasmic fragmentation was observed before the 4- to 8-cell stage of embryo development. Nuclear condensation was detected in morulae and blastocysts, but not all condensed nuclei were positive for the apoptotic signal (TUNEL staining). The mean blastocyst diameter increased with delayed blastocyst collection or extended post-blastulation culture, but decreased with delayed blastocyst formation. Delayed blastocyst collection/formation or an additional day of post-blastulation culture increased the frequencies of apoptosis, condensed nuclei, and low quality blastocysts (those showing a nuclear destruction that negated counting of the nuclei); increased the expression of the pro-apoptotic BAX gene; and reduced the ratio of ICM (inner cell mass) cells to TE (trophectoderm) cells. In addition, delayed blastocyst formation decreased POU5F1 gene expression. These results suggest that a delay in blastocyst collection/formation or an additional day of culture could increase the incidence of apoptosis, decrease the ICM:TE cell ratio, and influence the gene expression and diameter of blastocysts derived from in vitro-produced pig embryos. These findings provide a useful reference for improving the quality of in vitro-produced embryos.

Tauroursodeoxycholic acid enhances the development of porcine embryos derived from in vitro-matured oocytes and evaporatively dried spermatozoa

Evaporative drying (ED) is an alternative technique for long-term preservation of mammalian sperm, which does not require liquid nitrogen or freeze-drying equipment, but offers advantages for storage and shipping at ambient temperature and low cost. However, the development of zygotes generated from these sperms was poor. Here, we demonstrated that the supplementation of tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, during embryo culture improved the developmental competency of embryos derived from in vitro matured pig oocytes injected intracytoplasmically with boar ED spermatozoa by reducing the production of reactive oxygen species, the DNA degradation and fragmentation, and the expression of apoptosis-related gene Bax and Bak, and by increasing the transcription of anti-apoptosis gene Bcl-XL and Bcl-2. Furthermore, TUDCA treatment promoted the blastocyst quality manifested by the total cell numbers and the ratio of inner cell mass. Taken together, our data suggest that evaporative drying would be a potentially useful method for the routine preservation of boar sperm in combination with further optimization of subsequently embryo culture conditions.

Ratio between inner cell mass diameter and blastocyst diameter is correlated with successful pregnancy outcomes of single blastocyst transfers.

To evaluate the ability to predict pregnancy outcomes of single-blastocyst transfers by measuring the ratio of inner cell mass (ICM) diameter to blastocyst diameter using time-lapse images. Retrospective cohort study. University-affiliated medical center. One hundred twenty-seven women undergoing a total of 129 blastocyst transfers with intracytoplasmic sperm injection. Embryo monitoring by time-lapse microscopy. The ratio of ICM diameter to blastocyst diameter in single-blastocyst transfers and clinical pregnancy rates. In phase I of the study, 63 women underwent 65 single blastocyst transfers that resulted in 25 pregnancies (40% of the women). The successfully implanted blastocysts had an average ICM/blastocyst diameter ratio of 0.487 ± 0.086, whereas the average ICM/blastocyst ratio of nonimplanted blastocysts was significantly lower (0.337 ± 0.086). The live-birth rate was 29% (18/63). In phase II, 64 single-blastocyst transfers were performed in 64 women. The ICM/blastocyst diameter ratio was measured, and blastocysts with the highest ratios were chosen for transfer. Forty-three women (67%) with an average ICM/blastocyst diameter ratio of 0.46 achieved pregnancy, and 36 of the 43 pregnancies (84%) resulted in the delivery of a healthy baby. In the 21 women (33%) who failed to achieve pregnancy, the average ICM/blastocyst ratio was 0.45. The resultant positive predictive value was 74%, and the negative predictive value was 70%. The ICM-to-blastocyst diameter ratio is a predictor of implantation and live birth in single-blastocyst transfers, offering a simple, noninterfering method to select blastocysts with high developmental capacity.

Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo.

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm:ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01nM and expression of S100A10 at 1.0nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

Effects of Insulin-like Growth Factor-1 on Development of Somatic Cell Cloned Bovine Embryos

The aim of this study was to assess the effect of insulin-like growth factor-1 (IGF-1) on the developmental competence of somatic cell nuclear transfer (SCNT) bovine embryos. First, the expression levels of IGF-1 receptor (IGF-1R) and IGF-1 in the oocytes and embryos of different developmental stages were examined. Then the effects of exogenous IGF-1 on the development of SCNT embryos were evaluated both in vitro and in vivo. The results showed that IGF-1 was not expressed in both IVF and SCNT embryos, whereas IGF-1R could be detected throughout the preimplantation stages in both protein and mRNA levels. Also, exogenous IGF-1 had no obvious impact on the developmental competence of IVF embryos. However, it could improve the developmental competence of SCNT embryos in terms of blastocyst developmental rate (31.3% vs. 43.2%, p < 0.05), total cell number (93.0 ± 9.9 vs. 101.0 ± 9.8, p < 0.05), ratio of inner cell mass (ICM) to trophectoderm (TE) (0.29 ± 0.006 vs. 0.39 ± 0.005, p < 0.05), and apoptosis index in day 7 blastocysts (2.5 ± 0.22 vs. 8.7 ± 0.41, p < 0.05) compared to the control group. Although no statistical difference in pregnancy rate and birth rate was observed after embryo transfer, there was an upward tendency in both examined terms in the IGF-1-supplemented group when compared with the control group. In conclusion, the present study showed that supplementing exogenous IGF-1 to the culture medium has an obvious positive effect on the development competence of SCNT embryos.

93 DEVELOPMENT OF BOVINE PRE-IMPLANTATION EMBRYOS IS ALTERED BY ADDITION OF ACTIVIN AND CONNECTIVE TISSUE GROWTH FACTOR FROM DAYS 5 TO 7 AFTER FERTILIZATION

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. Here we tested actions of two molecules that are highly expressed in the endometrium for actions to modify development of bovine embryos. The molecules tested were activin and connective tissue growth factor (CTGF), and endpoints were percentage of fertilized oocytes becoming blastocysts and number of inner cell mass (ICM) and trophectoderm (TE) cells in Day 7 blastocysts. Bovine embryos produced in vitro from slaughterhouse ovaries were cultured in a serum-free culture medium. On Day 5 of culture, culture drops were supplemented with vehicle (control), human recombinant activin or CTGF (10–11, 10–10, or 10–9 M). On Day 7, blastocysts (n = 202) were collected and labelled with a nuclear dye (Hoescht 33342) and a TE cell marker (anti-CDX2). Statistical analysis was performed using the GLM procedure of SAS. Results are shown as least squares means ± s.e.M. The percentage of putative zygotes becoming a blastocyst on Day 7 was 28.4 ± 1.5% for control embryos; 32.2 ± 1.5%, 30.7 ± 1.5%, and 33.1 ± 1.5% for embryos cultured with 10–11, 10–10, and 10–9 M activin, respectively; and 27.8 ± 1.5%, 25.1 ± 1.5%, and 28.4 ± 1.5% for embryos cultured with 10–11, 10–10, and 10–9 M CTGF, respectively. Activin increased the proportion of putative zygotes becoming a blastocyst when added at 10–9 M (P = 0.0321) and tended to have the same effect at 10–11 M (P = 0.0898). There was no effect of any concentration of CTGF on development of putative zygotes to blastocyst on Day 7. There was no effect of treatment on total cell number or number of TE or of activin on ICM cell number or TE : ICM ratio. However, CTGF increased (P = 0.0011) the number of ICM (40.1 ± 1.5, 46.2 ± 1.4, 46.5 ± 1.4, and 56.3 ± 1.4 for control and 10–11, 10–10, and 10–9 M CTGF, respectively). CTGF also reduced (P = 0.0319) the TE : ICM ratio. Values were 2.8 ± 0.2 for control and 2.1 ± 0.2, 1.9 ± 0.2, and 1.6 ± 0.2 for 10–11, 10–10, and 10–9 M CTGF, respectively. Results indicate that activin and CTGF can affect embryo development. In particular, activin increases competence of embryos to develop to the blastocyst stage and CTGF affects blastocyst differentiation. Support was provided by NIH R03 HD080855.

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus-oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.

Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos.

This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P < 0.05). Our results showed that the addition of growth factors to IVM and sequential culture media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system.

Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) is a promising technology, but its application is hampered by its low efficiency. Hence, the majority of SCNT embryos fail to develop to term. In this study, the antioxidant melatonin reduced apoptosis and reactive oxygen species (ROS) in bovine SCNT embryos. It also increased cell number, inner cell mass (ICM) cell numbers, and the ratio of ICM to total cells while improving the development of bovine SCNT embryos in vitro and in vivo. Gene expression analysis showed that melatonin suppressed the expression of the pro-apoptotic genes p53 and Bax and stimulated the expression of the antioxidant genes SOD1 and Gpx4, the anti-apoptotic gene BCL2L1, and the pluripotency-related gene SOX2 in SCNT blastocysts. We also analyzed the epigenetic modifications in bovine in vitro fertilization, melatonin-treated, and untreated SCNT embryos. The global H3K9ac levels of melatonin-treated SCNT embryos at the four-cell stage were higher than those of the untreated SCNT embryos. We conclude that exogenous melatonin affects the expression of genes related to apoptosis, antioxidant function, and development. Moreover, melatonin reduced apoptosis and ROS in bovine SCNT embryos and enhanced blastocyst quality, thereby ultimately improving bovine cloning efficiency.

Effects of short-term exposure of mature oocytes to sodium nitroprusside on in vitro embryo production and gene expression in bovine

Stress tolerance can be induced in embryos by oocyte exposure to hydrostatic pressure, osmotic agents, heat shock, or reactive oxygen species. This study assessed the effects of exposing bovine oocytes to a nitric oxide (NO) donor, sodium nitroprusside (SNP), on subsequent in vitro embryo production, embryo quality and the expression of genes involved in NO production (iNOS, eNOS, and nNOS), stress tolerance (HSP70 and HSP90), oxidative stress (HIF1A and PRDX5), and apoptosis (BCL2A1). In vitro mature oocytes were incubated with SNP (control, 10−6M, 10−5M, and 10−4M) for 1 hour before in vitro fertilization, and cultured until Day 7. Cleavage and blastocyst rates were recorded. Next, embryo quality (ratio of inner cell mass to total cell number) and relative gene expression of iNOS, eNOS, nNOS, HSP70, HSP90, HIF1A, PRDX5, and BCL2A1 were determined in expanded blastocysts. Cleavage rates were significantly lower for 10−4M SNP compared with the control and 10−5M SNP treatments (77 ± 7.1%, 82 ± 8.4%, and 84.9 ± 4.1%, respectively). Total blastocyst rates were lower in the 10−4M SNP group relative to the control group (26.2 ± 4.9% and 34.1 ± 7.8%, respectively). Embryo quality was similar among the groups. However, our relative gene expression analysis revealed the downregulation of endothelial oxide nitric synthase messenger RNA in expanded blastocysts in all the treatment groups compared with the control treatment. These results suggest that the short-term exposure of mature bovine oocytes to a NO donor does not induce their stress tolerance and has no beneficial effect on the in vitro embryo production of bovine embryos.

The effect of macromolecule and growth factor combinations on in vitrodevelopment of bovine embryos

This study was conducted to determine the effects of different macromolecule sources added to synthetic oviduct fluid (SOF) culture medium supplemented with growth factors on the development of bovine embryos and blastocyst morphology. Zygotes were distributed into 5 treatment groups. Cleavage, morula, and blastocyst rates were evaluated under a stereomicroscope. Trophectoderm (TE) and inner cell mass (ICM) cells were determined by differential staining method. It was found that bovine serum albumin (BSA), either alone or in combination with growth factors, as compared to the control or polyvinyl-alcohol (PVA) resulted in higher embryo yield and faster development during early bovine embryo culture. The quality of bovine embryos, based on the number of blastocyst cells and the ratio of ICM to total blastomeres, was affected by the sources of macromolecules and their combinations with growth factors. Growth factors supplemented to SOFaa media with BSA and PVA significantly increased the number of ICM cells and the ratio of ICM cells to total number of cells. In conclusion, replacing BSA with PVA depressed the blastocyst rate and cell numbers, and the number of blastomeres and ICM and TE cell numbers were affected by both the type of macromolecule and the growth factor supplements.

Recombinant human growth differentiation factor-9 improves oocyte reprogramming competence and subsequent development of bovine cloned embryos.

Previously, we found that oocyte-secreted factors (OSFs) secreted by denuded oocytes during in vitro maturation (IVM) enhance subsequent development of bovine somatic cell nuclear transfer (SCNT) embryos. This treatment requires many oocytes during IVM. Hence, the aim of this study was to investigate whether supplementing with recombinant growth differentiation factor-9 (GDF9), one of crucial OFSs, in oocyte maturation medium could improve developmental competence of bovine oocytes and subsequent development of cloned embryos. Cumulus-oocyte complexes (COCs) from antral follicles of bovine ovaries collected from an abattoir were cultured with (SCNT+GDF9 group) or without (SCNT group) 200 ng/mL recombinant human GDF9 in oocyte maturation medium. After 22 h, metaphase II (MII) oocytes were used for SCNT. The presence of 200 ng/mL GDF9 significantly increased oocyte maturation rates, the cleavage rate, and blastocyst formation rates of bovine cloned embryos. The blastocyst total, inner cell mass (ICM) cell numbers, and ratio of ICM:TE were higher, whereas the rate of apoptosis in bovine cloned blastocysts was lower in the SCNT+GDF9 group than in the SCNT group. The histone modifications at various sites were also different between each group. These results suggest that COCs cultured with recombinant GDF9 in oocyte maturation medium improve oocyte developmental competence and subsequent developmental competence of cloned embryo in cattle.