Embryonic Development In Pigs Research Articles

Identification and characterization of L1-specific endo-siRNAs essential for early embryonic development in pig.

Small noncoding RNAs (sncRNAs) play important roles in RNA interference (RNAi). In addition to microRNA (miRNA) and Piwi-interacting RNA (piRNA), one key member of sncRNAs group is endogenous small interfering RNA (endo-siRNA). Some studies do show the role of endo-siRNAs in Dicer and/or Ago mutants, however, the biological functions of specific endo-siRNAs remains mostly unanswered. In the study, we have performed a comparative analysis of endo-siRNAs present in porcine sperms, oocytes and zygotes, identified by deep sequencing and bioinformatics analysis. Further, we observe a large amount of endo-siRNAs specific binding on ORF2 and 3′ UTR of porcine L1 (L1-siRNAs). And, 9 L1-siRNAs generated from a dsRNA formed between L1 transcript and a newly identified an antisense noncoding RNA was characterized. We show the L1-siRNAs regulate early embryonic development by inhibiting the activity of L1 retrotransposition. This work can contribute to understanding the functional role of abundant endo-siRNAs in embryonic development.

The influence and role of melatonin on in vitro oocyte maturation and embryonic development in pig and cattle

The influence and role of melatonin on in vitro oocyte maturation and embryonic development in pig and cattle

In vitro oocyte maturation in a medium containing reduced sodium chloride improves the developmental competence of pig oocytes after parthenogenesis and somatic cell nuclear transfer.

The present study investigated the effects of IVM in hypotonic medium containing reduced (61.6mM) NaCl compared with isotonic medium containing 108.0mM NaCl (designated L and N respectively) on oocyte maturation and embryonic development in pigs. IVM culture was divided into four periods at 11-h intervals. Oocytes cultured in N for 33h and then in L for 11h of IVM (N-N-N-L) showed significantly improved (P<0.05) nuclear maturation of oocytes (75.4-79.0% vs 60.2-85.8%) and blastocyst formation (61.5-66.1% vs 45.2-67.5%) after parthenogenesis (PA) compared with other treatments (L-L-L-L, L-L-L-N, L-L-N-L, N-N-L-L, N-N-L-N, L-L-N-L, L-N-N-L and N-L-N-L). Oocytes matured in L-L-L-L and N-N-N-L had an increased (P<0.05) perivitelline space (11.0-12.5 vs 5.5µm) and intraoocyte reduced glutathione (GSH) content (1.39-1.41 vs 1.00 pixels per oocyte) relative to oocytes matured in N-N-N-N. Somatic cell nuclear transfer (SCNT) embryos derived from the N-N-N-L treatment had significantly (P<0.05) higher blastocyst formation (53.5%) than embryos derived from Medium-199 (37.4%) and N-N-N-N (41.8%) treatments. Overall, the results demonstrate that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11h of IVM increases the developmental competence of oocytes after PA and SCNT by improving the cytoplasmic microenvironment, including an increased GSH content in IVM oocytes.

Inhibition of the Arp2/3 complex impairs early embryo development of porcine parthenotes

ABSTRACTThe Arp2/3 complex, which nucleates actin filaments, comprises a stable assembly of seven-protein subunits including two actin-related proteins (Arp2 and Arp3). Previous work showed that Arp2/3 binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. In the present study, we show that the Arp2/3 complex is critical for cytokinesis during early embryonic development in porcine parthenotes. The Arp2/3 complex is concentrated at the cortex of each cell at the 1-, 2-, and 4-cell stages, and at the periphery at the morula stage. The amount of Arp2/3 significantly decreased at the blastocyst stage in parthenogenetically activated porcine embryos. Inhibition of the Arp2/3 complex in the pig embryos by the Arp2/3-specific inhibitor CK666 resulted in abnormal cell division, a decrease in developmental rate and total cell numbers, and an increase in the ratio of trophectoderm cell number to inner cell mass number in blastocyst-stage embryos. In addition, 4-cell stage embryos subjected to CK666 treatment exhibited significantly decreased expression of ZGA genes (Pou5f1, Sox2, and Nanog), suggesting that the Arp2/3 complex plays an important role in early porcine embryo development. Thus, our data demonstrate that the Arp2/3 complex is required for early embryonic development in pigs and appears to regulate the expression of pluripotency genes.

Zinc deficiency during in vitro maturation of porcine oocytes causes meiotic block and developmental failure.

The present study investigated the effects of zinc deficiency during in vitro maturation (IVM) of porcine oocytes. Zinc deficiency was induced by administering the membrane‑permeable zinc chelator N,N,N',N'‑tetrakis‑(2‑pyridylmethyl)‑ethylendiamine (TPEN). First, the effects of zinc deficiency during IVM on a TPEN‑treated group and a TPEN+zinc-treated group compared with a control group were assessed. The oocyte maturation rates and subsequent embryonic developmental competence of the TPEN+zinc‑treated oocytes were similar to those of the control oocytes (metaphase II [MII] rate, 93.0 and 92.7%, respectively, and blastocyst [BL] formation rate, 42.0 and 40.0%, respectively). These results were significantly different from those obtained for the TPEN‑treated oocytes (MII rate, 0.61%; BL formation rate, 0%). Although the TPEN‑treated oocytes were arrested at metaphase I (MI), the distribution of microtubules was normal. However, microfilament formation was abnormal in the TPEN‑treated oocytes. Furthermore, the effect of a temporary zinc deficiency during IVM on oocyte maturation and subsequent embryonic development was assessed. TPEN (10 µM) was added to the IVM medium for 0, 7, 15 or 22 h. The 0 h‑treated oocytes showed an 83.9% MII rate, while the 7 h‑treated oocytes had a significantly lower MII rate (44.8%). Most of the 15- and 22 h‑treated oocytes were arrested at MI (MI rate: 98.0 and 97.2%, respectively; MII rate, 0% in both groups). Reductions in the BL formation were dependent on the TPEN treatment duration (29.3, 9.2, 0, and 0% after 0, 7, 15 and 22 h, respectively). In conclusion, zinc is an essential element for successful oocyte maturation and embryonic development in pigs. Zinc deficiency caused a meiotic block and had lasting effects on early embryonic development.

Effects of WT1 down-regulation on oocyte maturation and preimplantation embryo development in pigs.

The Wilms' tumour 1 (WT1) gene originally identified as a tumour suppressor associated with WTs encodes a zinc finger-containing transcription factor that is expressed in multiple tissues and is an important regulator of cellular and organ growth, proliferation, development, migration and survival. However, there is a deficiency of data regarding the expression and function of WT1 during oocyte maturation and preimplantation embryonic development. Herein, we sought to define the expression characteristics and functions of WT1 during oocyte maturation and preimplantation embryonic development in pigs. We show that WT1 is expressed in porcine oocytes and at all preimplantation stages in embryos generated by ICSI. We then evaluated the effects of down-regulating WT1 expression at germinal vesicle and early ICSI stages using a recombinant plasmid (pGLV3-WT1-shRNA). Down-regulation of WT1 did not affect oocyte maturation but significantly decreased preimplantation embryonic development and increased apoptosis in blastocysts. These results indicate that WT1 plays important roles in the development of porcine preimplantation embryos.

Epigenetic modification agents improve genomic methylation reprogramming in porcine cloned embryos.

Incomplete DNA methylation reprogramming in cloned embryos leads to low cloning efficiency. Our previous studies showed that the epigenetic modification agents 5-aza-2’-deoxycytidine (5-aza-dC) or trichostatin A (TSA) could enhance the developmental competence of porcine cloned embryos. Here, we investigated genomic methylation dynamics and specific gene expression levels during early embryonic development in pigs. In this study, our results showed that there was a typical wave of DNA demethylation and remethylation of centromeric satellite repeat (CenRep) in fertilized embryos, whereas in cloned embryos, delayed demethylation and a lack of remethylation were observed. When cloned embryos were treated with 5-aza-dC or TSA, CenRep methylation reprogramming was improved, and this was similar to that detected in fertilized counterparts. Furthermore, we found that the epigenetic modification agents, especially TSA, effectively promoted silencing of tissue specific genes and transcription of early embryo development-related genes in porcine cloned embryos. In conclusion, our results showed that the epigenetic modification agent 5-aza-dC or TSA could improve genomic methylation reprogramming in porcine cloned embryos and regulate the appropriate expression levels of genes related to early embryonic development, thereby resulting in high developmental competence.

Supplementation of insulin–transferrin–selenium to embryo culture medium improves the in vitro development of pig embryos

Insulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

Knockdown of Maternal Homeobox Transcription Factor SEBOX Gene Impaired Early Embryonic Development in Porcine Parthenotes

A number of germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, the role of these factors during early embryonic development has been poorly explored. In the present study, we investigated the role of SEBOX, a maternal homeobox transcription factor, during early embryonic development in porcine parthenotes. mRNA for SEBOX is preferentially expressed in oocytes, and expression persists until embryonic genome activation (EGA). Knockdown of SEBOX by siRNA disrupted early embryonic development, but not oocyte maturation. Many maternal genes essential for early embryonic development were upregulated in SEBOX-depleted embryos. Moreover, some pluripotency-associated genes, including SOX2 and NANOG, were upregulated when SEBOX was knocked down. Therefore, our data demonstrate that SEBOX is required for early embryonic development in pigs and appears to regulate the degradation of maternal transcripts and the expression of pluripotency genes.

Leptin and nonessential amino acids enhance porcine preimplantation embryo development in vitro by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been considered one of the strong assisted reproductive technologies for producing transgenic animals as well as treating infertility in animals and humans. However, in porcine ICSI, embryos produced by in vitro methods show low pregnancy rates with high abnormal offspring and blastocyst formation rate as well as quality are poor compared with those in other species. For these reasons, developing a protocol for porcine ICSI is essential to efficiently generate transgenic pigs. Since amino acids were introduced to embryo development because of their beneficial effects, many embryologists have been using nonessential amino acid (NEAA) in culture medium for embryonic development in pig and other species. Leptin also has been shown to be beneficial in embryonic development for increasing rate of cleavage and blastocyst development. However, the effects of NEAA and leptin were not fully understood in the development of porcine ICSI-derived embryos. Here we investigated the optimization of NEAA and leptin supplementation in culture medium to improve developmental competence and quality of preimplantation embryos after ICSI in pig. The proportion of embryos that developed to the blastocyst stage was significantly greater when 1% vol/vol NEAA (24.6%) or 100 ng/mL leptin (27.1%) was supplemented in the culture medium compared with other concentrations or no supplement. When NEAA and leptin (24.8%) were supplemented together, blastocyst formation was significantly higher than other single supplementation groups. We also evaluated the effects of different supplementation periods of NEAA or leptin on the preimplantation embryonic development after ICSI. Both NEAA and leptin showed that supplementation for the entire 7 days significantly increased the blastocyst formation rate compared with the other groups of supplementation for the first 4 days and for the subsequent 3 days. A second goal of our research was to evaluate the quality of developed blastocysts after ICSI. The supplementation of 100 ng/mL leptin in culture medium made blastocysts express less of the proapoptosis genes BAX and BAK and more of the antiapoptosis genes BCL-XL and BCL-2 after the ICSI procedure. Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased when the ICSI-derived embryos were cultured to blastocyst stage in the presence of the combination of NEAA and leptin. These results suggest that NEAA and leptin could improve not only the quantity but also quality of ICSI-derived porcine embryos during in vitro culture with the optimal concentration of each reagent.

Identification and Characterization of Pig Embryo MicroRNAs by Solexa Sequencing

MicroRNAs (miRNAs) are a class of small, non-coding RNAs of approximately 22 nucleotides in length that regulate gene expression by binding to the 3'-untranslated regions of target mRNAs. It is now clear that miRNAs are involved in many biological processes, including proliferation, differentiation and regulation of gene expression during early embryonic development. The miRBase 16.0 (2010) shows that there are 175, 673, 408 and 1048 annotated miRNAs for Caenorhabditis elegans, Mus musculus, Rattus norvegicus and Homo sapiens, respectively. However, there are only 211 miRNAs described for Sus scrofa. In particular, the full set of miRNAs and their expression patterns are still poorly understood in the embryo. Therefore, we combined Solexa sequencing with computational techniques to analyse the sequences and relative expression levels of S. scrofa miRNAs at embryonic day 33 (E33). Of the distinct miRNAs identified, 76 previously known miRNAs and 194 candidate miRNAs were identified in head, and 77 known miRNAs and 130 predicted candidate miRNAs were identified in organ region. Furthermore, we performed additional investigation for identifying the potential target mRNAs using PicTar and TargetScan. Concurrent function analysis suggested that highly expressed miRNAs are mostly involved in the development of nerves, cerebrum, muscle and organs. Our results provide useful information for the investigation into embryonic miRNAs of pig and provide a valuable resource for investigators interested in the regulation of embryonic development in pigs and other animals.

Iloprost, a prostacyclin analogue, stimulates meiotic maturation and early embryonic development in pigs

Oviduct fluid contains various cytokines and growth factors that enhance the embryo development during the preimplantation period. In hatched embryos, prostacyclin (PGI(2)) improves implantation, but its role during oocyte maturation and early embryo development remains contentious. Therefore, in the present study, we examined the effects of a PGI(2) analogue (iloprost) on meiotic maturation and early embryonic development in pigs, as well on the structural integrity, mitochondrial membrane potential and apoptosis in blastocysts. First, meiotic maturation in pig oocytes was examined in the presence of increasing concentrations of iloprost (1, 5 and 10 muM). After IVM, a higher proportion of iloprost-treated compared with untreated oocytes was in MII (90.0% v. 65.7%, respectively; P < 0.05). In addition, protein kinase A activity increased in iloprost-treated oocytes, indicating increased intracellular cAMP concentrations. After 22 h iloprost treatment (44 h total incubation time), western blotting demonstrated increased expression of extracellular signal-regulated kinase (ERK) 1/2, phosphorylated (p-) ERK1/2, cAMP response element-binding protein (CREB), p-CREB and cyclo-oxygenase-2, indicating activation of the mitogen-activated protein kinase and PGI(2) pathways. In addition, the frequency of polyspermy decreased in iloprost-treated oocytes (19.9%) compared with control (35.8%), whereas the rate of blastocyst formation increased (P < 0.05). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) showed that the number of nuclei containing fragmented DNA at the blastocyst stage decreased in the iloprost-treated group compared with control (1.2% v. 3.6%, respectively). In conclusion, iloprost appears to play a direct role in porcine oocyte maturation by enhancing blastocyst structure and survival.

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO 2 in air for 40–44 h. No significant difference ( P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased ( P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced ( P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser ( P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced ( P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.

Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation; it stimulates further embryonic development including blastocyst quality in pigs

High concentrations of cyclic AMP in germinal vesicle oocytes generally inhibit GVBD. Thus, maintaining the GV stage in growing oocytes is essential for the developmental competence of the eggs. In this study, we traced the effects of dibutyryl cyclic AMP on meiotic maturation and early embryonic development in pigs. We also investigated several blastocyst qualities, including structural integrity, mitochondrial membrane potential, and apoptosis, which are affected by dbcAMP. To determine whether increased concentrations of cAMP inhibit GVBD, we explored the meiotic patterns and during maturation of pig oocytes. When treated with dbcAMP for 22h, 91.1% of the oocytes were arrested in the GV stage compared to only 38.8% of the oocytes in the control group (P<0.05). After completion of IVM, a higher proportion of the dbcAMP-treated oocytes were in metaphase II than the untreated ones (91.3% vs. 72.8%, P<0.05). Western blot analysis showed a reduction (at 22h) and/or increase (at 44h) in MPF and MAP kinase activities in porcine oocytes treated with dbcAMP for the first 22h of IVM compared to the untreated control. We also confirmed that protein kinase A activity increased in dbcAMP-treated oocytes, indicating an elevated intracellular concentration of cAMP. After IVF, the frequency of polyspermy in the dbcAMP-treated group decreased compared to that in the control group (22.4% vs. 47.4%, P<0.05). Furthermore, blastocyst formation, the blastocyst cell number, mitochondrial membrane potential, and apoptosis were enhanced and/or reduced by dbcAMP in both IVF and SCNT embryos. We concluded that synchronizing meiotic resumption by dbcAMP treatment improved the developmental capacity and embryonic qualities of IVF and SCNT embryos by increasing the mitochondrial membrane potential and decreasing the incidence of apoptosis in preimplantation-stage porcine embryos.

Ultrastructural study of polyspermy during early embryo development in pigs, observed by scanning electron microscope and transmission electron microscope.

Polyspermy is generally considered a pathological phenomenon in mammals. Incidence of polyspermy in porcine eggs in vivo is extremely high (30-40%) compared with other species, and polyspermy rate in the in vitro fertilized eggs in pigs can reach 65%. It is still unknown whether polyspermy to a certain degree is a physiological condition in pigs, and whether porcine eggs have any capability with which to remove the accessory sperm in the cytoplasm. The objectives in the present study are to observe the ultrastructural changes of accessory sperm during early embryonic development in pigs. A total of 58 normal, early embryos at one-, two, three-, and four-cell and morular stages were collected from gilts and were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface ultrastructure showed that sperm fusion with the zona pellucida was a continuous process during one-, two-, three-, and four-cell and morular stages, as observed by the SEM. Accessory sperm were present in the cytoplasm of cleaved embryos. The sperm heads in the cytoplasm of cleaved embryos did not decondense. TEM revealed the presence of a condensed sperm head within a lysosome (or phagolysosome) in a three-cell embryo. These observations suggest that polyspermy may be a physiological condition in pigs and that early embryos may develop to term if accessory sperm do not interrupt the embryo genome. Furthermore, lysosome activity could be another physiological mechanism for removing accessory sperm in the cytoplasm of fertilized eggs and cleaved embryos after fertilization in pigs.

Expression of glycine cleavage system and effect of glycine on in vitro maturation, fertilization and early embryonic development in pigs

Glycine is the amino acid with the highest concentration in sow oviduct fluid, uterine fluid and follicular fluid. In this study, the effect of glycine on in vitro maturation, fertilization and early embryonic development in pigs was evaluated. In addition, the expression of T-protein, a component of the glycine cleavage enzyme system, was studied in the oocytes, cumulus cells, granulosa cells and oviductal epithelial cells using the methods of reverse transcription-polymerase chain reaction (RT-PCR). The results showed that supplementation of porcine oocytes with glycine during in vitro maturation improved on the development of embryos in vitro. After fertilization, glycine also enhanced embryo cleavage rate in the presence, but not in the absence, of oviductal cells. PCR product corresponding to T-protein was found in uncultured oocytes, cumulus cells, granulosa cells and oviductal epithelial cells. These results indicate that glycine is an important amino acid for the in vitro oocyte maturation, fertilization and early embryonic development in pigs.