Parthenogenetic Embryos Research Articles

Optimizing camel (Camelus dromedarius) oocytes in vitro maturation and early embryo culture after parthenogenetic activation

This study was undertaken to investigate on camel oocytes the effects of different in vitro maturation (IVM) time, supplementation of the IVM medium on oocyte maturation and the use of different in vitro culture media (KSOMaa, mSOF, TCM, and K-SICM) on embryo development to optimize in vitro embryo production procedures. Cumulus-oocyte complexes (COCs) were cultured for 28, 32, 36, 42, and 48h for IVM and were examined for cumulus expansion, meiotic maturation, and early embryo development after parthenogenetic chemical activation and IVC. The results showed that IVM of oocytes for 32 and 36h significantly increased embryonic development (P<0.05) compared with other treatments. Culturing the oocytes for longer durations (42 and 48h) permitted the entry and completion of meiosis II and significantly reduced embryonic developmental competence, as indicated by reduction in morula and blastocyst ratios (P <0.05). Moreover, supplementation of IVM medium with follicular fluid improved cumulus expansion when compared with fetal bovine serum supplementation. Culturing of parthenogenetic embryos in KSOMaa medium significantly improved morula and blastocyst development, compared with culturing in mSOF, TCM, and K-SICM media (P<0.05). We conclude that IVM of camel oocytes for a period of 32–36h would be sufficient to support early embryonic development after parthenogenetic activation and that KSOMaa is an efficient culture medium for in vitro camel embryo production.

Identification of brain proteins BASP1 and GAP-43 in mouse oocytes and zygotes

The similarity between the calcium-activated signaling systems of oocytes and neuronal axon terminals has prompted us to test whether BASP1 and GAP-43 proteins, highly expressed in brain neurons, are present in oocytes. Using immunocytochemical techniques combined with confocal microscopy, we have for the first time demonstrated that both BASP1 and GAP-43 are present in mouse metaphase II (MII) oocytes and zygotes. BASP1 is localized to the plasma membrane and actin cortex of MII oocytes, which is similar to BASP1 distribution in neurons and other cell types. GAP-43 is generally regarded as a postmitotic membrane marker of nerve cells; however, GAP-43 in MII oocytes is associated with microtubules of the meiotic spindle. GAP-43 is also colocalized with γ-tubulin at the spindle poles (centrosomes) and at the discrete microtubule- organizing centers in the cytoplasm. The antibodies to Ser41-phosphorylated form of GAP-43 allowed for demonstration that GAP-43 in oocytes is subject to phosphorylation by protein kinase C. The presence of BASP1 and GAP-43 in oocytes is also confirmed by electrophoresis and western blotting. Microinjection of BASP1 (but not GAP-43) into the cytoplasm of mouse MII oocytes induces their exit from metaphase II arrest followed by parthenogenetic embryo development. This suggests putative BASP1 involvement in fertilization-induced oocyte activation, presumably, through regulation of local concentration of polyphosphoinositides in the plasma membrane. Recently it was found that GAP-43 is associated with centrosomes in asymmetrically dividing neuronal progenitors, which is similar to the localization of GAP-43 at the meiotic spindle and centrosomes in oocytes. Therefore we suggest that GAP-43 may be involved in regulation of spindle orientation and oocyte polarity.

Generation of chimeric minipigs by aggregating 4- to 8-cell-stage blastomeres from somatic cell nuclear transfer with the tracing of enhanced green fluorescent protein.

Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young. Here, we used early-stage embryos from somatic cell nuclear transfer (SCNT) to generate chimeric piglets by the aggregation method. Then, the distribution of chimerism in various tissues and organs was observed through the expression of enhanced green fluorescent protein (EGFP). Initially, we determined whether 4- to 8- or 8- to 16-cell-stage embryos were more suitable to generate chimeric piglets. Chimeras were produced by aggregating two EGFP-tagged Wuzhishan minipig (WZSP) SCNT embryos and two Bama minipig (BMP) SCNT embryos. The chimeric piglets were identified by coat color and microsatellite and swine leukocyte antigen analyses. Moreover, the distribution of chimerism in various tissues and organs of the piglets was evaluated by EGFP expression. We found that more aggregated embryos were produced using 4- to 8-cell-stage embryos (157/657, 23.9%) than 8- to 16-cell-stage embryos (100/499, 20.0%). Thus, 4- to 8-cell-stage embryos were used for the generation of chimeras. The rate of blastocysts development after aggregating WZSP with BMP embryos was 50.6%. Transfer of 391 blastocysts developed from 4- to 8-cell-stage embryos to five recipients gave rise to 18 piglets, of which two (11.1%) were confirmed to be chimeric by their coat color and microsatellite examination of the skin. One of the chimeric piglets died at 35days and was subsequently autopsied, whereas the other piglet was maintained for the following observations. The heart and kidneys of the dead piglet showed chimerism, whereas the spinal cord, stomach, pancreas, intestines, muscle, ovary, and brain had no chimerism. To our knowledge, this is the first report of porcine chimeras generated by aggregating 4- to 8-cell-stage blastomeres from SCNT. We detected chimerism only in the skin, heart, and kidneys. Collectively, these results indicate that aggregation using 4- to 8-cell-stage SCNT embryos offers a practical approach for producing chimeric minipigs. Furthermore, it also provides a potential platform for generating interspecific chimeras between pigs and non-human primates for xenotransplantation.

Endoplasmic Reticulum (ER) Stress and Apoptosis in Parthenogenetic Porcine Embryos following Different Combination of Activation Methods

Endoplasmic Reticulum (ER) Stress and Apoptosis in Parthenogenetic Porcine Embryos following Different Combination of Activation Methods

Effects of melatonin on maturation, histone acetylation, autophagy of porcine oocytes and subsequent embryonic development.

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle-derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.

Non-coding RNAs: emerging regulatory factors in the derivation and differentiation of mammalian parthenogenetic embryonic stem cells.

Parthenogenetic embryonic stem cells (PESCs) are ESCs derived from early parthenogenetic embryos. Haploid PESCs, containing haploid DNA, originate from a single sperm or occyte, while, diploid PESCs originate from two fused occytes. Most PESC lines used so far are diploid. PESCs exhibit representative pluripotent stem cell features, such as the capacity for self-renewal and the pariticular molecular signatures. Whereas, PESCs display distinctive properties, such as differential regulation of X-chromosome inactivation (XCI) and divergent monitor of genes involved in multiple biological processes. PESCs are considered promising in the regeneration medicine and developmental biology. Non-coding RNAs (ncRNAs), especially miRNAs and lncRNAs, have garnered increasing attention over the past 2 decades. They are now known to be involved in almost all cellular processes due to their full-range regulation of gene expression. Numerous studies have indicated that embryonic stem cells (ESCs) displayed distinct signatures of ncRNA genes, which play key roles in the pluripotency and self renewal of ESCs. However, the expression pattern of ncRNAs in PESCs and their roles in the derivation and differentiation of PESCs were rarely reported. In this paper, we reviewed recent research on the derivation and differentiation of PESCs and describe the emerging role of ncRNAs in these processes.

The effects of genotypes and irradiation doses on haploid embryo induction and plant production in bottle gourd [Lagenaria siceraria(Malign) Stanley

Cultivated Legenaria siceraria (Malign) Stanley is commonly known as the white-flowered gourd. It has been grown annually and it is a monocious, vigorous climber species. The mature fruit is often scooped out and the skin is used as containers, music instrument, decorative purposes or in some cases, fishing floats. Shoots, tendrils and leaves are also cooked and the seeds are removed for oil extraction or for use in cooking. Furthermore, L. siceraria is used as rootstocks conferring the watermelon grafted plant resistance to soil-borne diseases and adverse soil conditions. The objective of this study was to optimize bottle gourd haploid plants. Haploid plants are an invaluable source to rapidly produce new homozygous inbred lines. We analyzed the effect of various pollen irradiation doses (50, 75, 100, 125, 150, 175 and 200 Gray gamma rays of 60Co) on the induction of parthenogenetic haploid embryos after pollination with the irradiated pollen in four bottle gourd landraces (Adana, Birecik, Spain and Cyprus). Our results indicate that pollination with irradiated pollens at 50 and 75 Gy had a positive effect on haploid embryo induction. After pollination with irradiated pollens, embryos with different shape were rescued, cultured and converted to intact plants under tissue culture conditions. The genotypes had important effect on induction of haploid embryo by irradiated pollen technique. Seventy five plants from Adana genotype, 10 plants from Birecik genotype, 121 plants from Spain genotype, 141 plants from Cyprus genotype and totally 347 plants were produced from this research.

Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle

The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency.

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

Effects of Alcohol on Mitochondrial Functions of Cumulus Cells in Mice.

Alcohol is an important compound used in food, agriculture, and medicine. In this study, we investigated the effect of alcohol on oocyte quality in mice by exposing animals for different duration times during an estrous cycle. Cumulus-oocyte complexes were collected from mice after pregnant mare serum gonadotropin- and human chorionic gonadotropin-induced superovulation. Ovulation number, E2 level in serum, and parthenogenetic embryo development in vitro were evaluated. Mitochondrial gene expression, mitochondrial membrane potential, and reactive oxygen species (ROS) levels in the cumulus were also assessed. The results showed that acute exposure to alcohol did not affect ovulation time (p > 0.05). Blasocyst formation rate in vitro was significantly improved after 1 and 2 days of alcohol exposure (p < 0.01). Mitochondrial membrane potential was significantly increased after 1-4 days of alcohol exposure (p < 0.05), but it decreased after 5 days (p < 0.05). ROS levels remained relatively low after 2, 3, and 4 days of exposure (p < 0.05), and they significantly increased after 6 days (p < 0.05). In addition, alcohol altered the expression of mitochondrial and nuclear genes in the cumulus. Taken together, our data suggest that acute exposure to alcohol affects oocyte quality by influencing the function and gene expression in the cumulus. These results underscore potential implications for the development of human reproductive therapeutics.

Plk1 inhibition leads to a failure of mitotic division during the first mitotic division in pig embryos.

This study was conducted to examine the dynamic distribution of polo-like 1 kinase (Plk1) and the possible role it plays in first mitotic division during early porcine embryo development. Indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses were used to study the dynamic expression and subcellular localization of Plk1 protein in pig parthenogenetic embryos. Finally, a selective Plk1 inhibitor, GSK461364, was used to evaluate the potential role of Plk1 during this special stage. The results showed that Plk1 upon expression exhibited specific dynamic intracellular localization, which closely correlated with the α-tubulin distribution during the first mitotic division. GSK461364 treatment resulted in cleavage failure, with majority of the GSK461364-treated embryos being arrested in prometaphase. Further results of the subcellular structure examination showed that GSK461364 treatment led to a significantly higher proportion of the treated embryos having abnormal spindles and misarranged chromosomes at the prometaphase stage. Thus, these results indicated that Plk1 is essential for porcine embryos to complete the first mitotic division. Furthermore, Plk1 regulation was associated with effects on spindle assembly and chromosome arrangement.

Aggregation of a parthenogenetic diploid embryo and a male embryo improves the blastocyst development and parthenogenetic embryonic stem cell derivation

Parthenogenetically derived mammalian embryos, with no paternal genome, are not viable and die, largely from defective placental growth attributed to a lack of paternal effect, resulting in the low blastulation rate and low derivation efficiency of parthenogenetic embryonic stem cells (pESCs). Therefore, the present study, by the optimization of parthenogenetic embryo production and the aggregation of the parthenogenetic diploid embryo and the identified male embryo, aims to investigate a method to improve the development of parthenogenetic embryo and pESC derivation in mice. Using different chemical combinations for the optimization, we found that the heterozygous diploid type had a significantly higher blastulation rate than the haploid type (P < 0.05). The treatment of strontium chloride (SrCl2) combined with cytochalasin B for 4 h produced the highest heterozygous diploid rate and blastulation rate. Our self-made concave hole system was used for the aggregation of the parthenogenetic heterozygous diploid embryo with the male embryo identified by the duplex PCR method, and we found that the chimeric embryo had an improved rate of blastulation and pESC isolation.

Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts.

Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.

The effect of poly(ADP-ribosyl)ation inhibition on the porcine cumulus-oocyte complex during in vitro maturation

Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos.

85 REGULATION OF STEAROYL-CoENZYME A DESATURASE BY FATTY ACIDS IS ESSENTIAL TO PORCINE EARLY EMBRYO DEVELOPMENT

Various fatty acids are found in large amounts in follicular fluid and the uterus. These fatty acids are known to regulate lipid metabolism in a mammal’s embryo. Lipid metabolism provides a source of energy, and its importance during embryogenesis is being increasingly recognised. Especially, the pig has larger amounts of intercellular lipid bilayers in the embryo than do other species, which indicates the porcine embryo is more dependent on fatty acid on their metabolic pathway. This study investigated the transcriptome analysis data of in vivo embryos and the effect of oleic acid (C18:1n-9) and stearic acid (C18:0) on in vitro-produced porcine embryos. In transcriptome analysis of in vivo embryos, we found several genes were increasing before and after maternal zygotic transition stage, and interactions were mapped and given a significance score. Among these genes, stearoyl-coenzyme A desaturase (SCD) gene was significantly increased during 8-cell to blastocyst stage. Stearoyl-coenzyme A desaturase is responsible for converting saturated fatty acid (stearic acid) to monounsaturated fatty acid (oleic acid). Furthermore, we treated with oleic acid (50, 100, 250, and 500 μM) and stearic acid (50, 75, and 100 μM) on 2 day and 4 day after parthenogenetic embryos. Both oleic acid and stearic acid concentration over 100 μM had a negative effect on blastocyst formation rate and cell numbers because of exogenous fatty acid toxicity. In addition, there was no significant difference among all stearic acid treated groups and total cell number of oleic acid treated blastocyst. However, the 2-day oleic acid treated group (45.92 ± 4.01, n = 8, 2 day, 100 μM) had a significant increase of blastocyst formation rate (P &lt; 0.05) compared with the control group (34.88 ± 2.93, n = 8). These data could support that porcine embryos can use exogenous oleic acid as a metabolic energy source. The data also demonstrate the important role of SCD in porcine early embryo development. To confirm the transcriptome analysis, we are investigating mRNA level of SCD on in vitro-produced embryos at 1-cell to morula and blastocyst stage on the control group, oleic acid treated group, fetal bovine serum treated group, and nontreated group. Furthermore, we are investigating SCD inhibition assay by A939572 (SCD inhibitor) on parthenogenetic embryos in a fetal bovine serum treated group and nontreated group for identifying blastocyst formation rate and total cell numbers. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2055199) and Next BioGreen 21 program (PJ0113002016), Rural Development Administration, Republic of Korea.

168 THE DEVELOPMENTAL POTENTIAL OF PARTHENOGENETIC EMBRYOS IS AFFECTED BY PROLACTIN DURING THE PROLONGED CULTURE OF BOVINE CUMULUS-ENCLOSED OOCYTES

The technique of somatic cell nuclear transfer includes a delayed activation of reconstituted oocytes (4–6 h after IVM). The prolonged culture of mammalian oocytes is known to be associated with aging of the ova leading to a decline in their quality and developmental capacity. The aim of the present research was to study effects of 2 closely related hormones, prolactin (PRL) and growth hormone (GH), during the prolonged culture of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-enclosed oocytes (CEO) were cultured for 16 h in TCM 199 containing 10% FCS, 10 μg mL−1 of porcine FSH, and 10 μg mL−1 of ovine LH. After 16 h of maturation, CEO or denuded oocytes were transferred to the fresh medium consisting of TCM 199 supplemented with 10% FCS and cultured for 12 h in the absence (Control) or in presence of 50 ng mL−1 of bovine PRL or 10 ng mL−1 of recombinant bovine GH or protein kinase inhibitors. The following inhibitors were used: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 10 mM), (2) triciribine (an inhibitor of Akt kinase, 25 μM), and (3) calphostin C (a protein kinase C inhibitor, 0.5 μM). After the prolonged culture for 12 h, oocytes were activated by sequential treatment with ionomycin (5 μM for 5 min) immediately followed by 6-dimethylaminopurine (2 mM for 4 h). Activated oocytes were cultured in CR1aa medium until Day 5 postactivation and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 7. The cleavage and blastocyst rates were assessed at Day 2 and 7, respectively. The data from 4 to 6 replicates (106–169 oocytes per treatment) were analysed by ANOVA. After the prolonged culture of CEO, the cleavage rate did not differ between the control and the hormone-treated groups, varying from 64.3 to 70.1%. Meanwhile, the blastocyst yield in the control group (12.5 ± 1.6%) was lower than in the PRL group (21.5 ± 2.4%, P &lt; 0.05), but was similar to that in the GH group (13.9 ± 1.8%). Furthermore, the developmental capacity of cultured denuded oocytes was unaffected by both GH and PRL. The enhancing effect of PRL on the yield of parthenogenetic blastocysts derived from CEO was also observed in the presence of PP2 (26.8 ± 2.4 v. 15.7 ± 2.0%; P &lt; 0.01). By contrast, calphostin C abolished this effect of PRL, although it did not affect the developmental potential of CEO in the control medium. At the same time triciribine reduced the blastocyst rate both in the control and PRL-treated groups (from 16.5 ± 1.8 to 8.1 ± 0.8% and from 24.3 ± 1.6 to 16.3 ± 2.4%, respectively; P &lt; 0.05). Our data indicate that PRL can support the developmental potential of parthenogenetic embryos by affecting bovine oocytes during the prolonged culture, with the effect being mediated by cumulus cells and achieved via activation of protein kinase C. This research was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 15-08-99473).

90 OVIDUCTAL EPITHELIAL CELLS Co-CULTURE PROMOTES GOAT (CAPRA HIRCUS) IN VITRO PARTHENOGENETIC EMBRYO DEVELOPMENT

Co-culture of pre-implantation embryos with oviducal epithelial cells mimics the in vivo conditions, thus, playing a crucial role in embryo metabolism and gene expression and finally supporting embryonic developmental competence in several ways. Hence, the objective of the present study was to evaluate the effect of goat oviducal epithelial cells (GOEC) co-culture on goat parthenogenetic embryonic development, quality, and relative mRNA abundance of genes related to developmental competence and oxidative stress. The GOEC were obtained from goat oviducts by squeezing and thorough washing with TCM-199 + 10% fetal bovine serum. Goat cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured in TCM-199 + 10% fetal bovine serum supplemented with 5 μg mL−1 of FSH, 10 μg mL−1 of LH, and 1 μg mL−1 of β-oestradiol for 27 h in CO2 incubator with 5% CO2 and at 38.5°C with &gt;95% RH. In vitro matured cumulus–oocyte complexes were denuded and activated with 5 μM calcium ionophore and 2 mM 6-DMAP. Following activation, embryos were co-cultured with and without GOEC (control) in mCR2aa media. The blastocyst development rate was significantly (P &lt; 0.05) higher (23.00 ± 1.15% v. 17.33 ± 1.45%) in the media cultured with GOEC than in control. The total cell number of blastocysts (n = 4) was also found to be significantly more (167.25 ± 17.51 v. 110.25 ± 12.02) than that of control (P &lt; 0.05). However, the apoptotic index (3.76 ± 0.23% v. 7.97 ± 1.99%) was not significantly different in both groups. Further, RNA was isolated from both groups (20 each) of blastocysts on Day 8, and cDNA was prepared. Analysis by qPCR revealed that the relative mRNA abundance of development related genes, i.e. VEGF, BMP4, and CCNB1, showed significantly high (P &lt; 0.05) expression, whereas the expression of CRABP1 was significantly low (P &lt; 0.05) in GOEC co-culture than control. Oxidative stress related genes GPX-1 and SOD2 had comparable expression in both the culture systems, whereas a nonsignificant (P &lt; 0.05) increase in expression of PRDX1 was observed in GOEC co-culture group. In conclusion, co-culture of embryos with GOEC in the simple culture media like mCR2aa helps in improving developmental competence and quality of parthenogenetic embryos. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.

Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice.

In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.

Improved development by melatonin treatment after vitrification of mouse metaphase II oocytes

The study was aimed to investigate the effect of melatonin on the development potential of mouse MII oocytes after cryopreservation. Mouse MII oocytes were subjected first to vitrification/warming and 2 h of in vitro culture (phase 1), then to parthenogenetic activation (PA) followed by in vitro culture of parthenogenetic embryos (phase 2). Different concentrations of melatonin (0, 10−9, 10−6 mol/L) were added to the medium during either phase 1, phase 2 or both phases. The fresh oocytes were used as control. When melatonin was used during both phases, 10−9 mol/L melatonin-treated group showed similar rates of cleavage and 4-cell embryo development compared with control, which were significantly higher than those of melatonin-free group, while the rates in either 10−6 mol/L melatonin-treated or melatonin-free groups were significantly lower than that in control. When 10−9 mol/L melatonin was added during either phase 1 or phase 2, both cleavage and 4-cell embryo development rates of either group were significantly lower than those of control. After oocyte vitrification/warming and PA, the ROS levels increased significantly and maternal-to-zygotic transition (MZT) related genes (Dcp1a, Dcp2, Hspa1a, Eif1ax, Pou5f1, Sox2) expression were disorganized. However, after 10−9 mol/L melatonin supplementation, the ROS levels decreased significantly compared with melatonin-free group, and the gene expressions were almost recovered to normal level of control group. These results demonstrated that 10−9 mol/L melatonin supplementation could increase the developmental potential of vitrified-warmed mouse MII oocytes, which may result from ROS scavenging activities and recovery of normal levels of the expressions of MZT-related genes.

Determination of timing and mechanism of diploidization of haploid parthenogenetic and androgenetic human embryos

Determination of timing and mechanism of diploidization of haploid parthenogenetic and androgenetic human embryos