Preimplantation Ovine Embryos Research Articles

Global proteomic analysis of preimplantational ovine embryos produced in vitro.

The present study was conducted to characterize the major proteome of preimplantation (D6) ovine embryos produced in vitro. COCs were aspirated from antral follicles (2-6mm), matured and fertilized in vitro and cultured until day six. Proteins were extracted separately from three pools of 45 embryos and separately run in SDS-PAGE. Proteins from each pool were individually subjected to in-gel digestion followed by LC-MS/MS. Three 'raw files' and protein lists were produced by Pattern Lab software, but only proteins present in all three lists were used for the bioinformatics analyses. There were 2,262 proteins identified in the 6-day-old ovine embryos, including albumin, zona pellucida glycoprotein 2, 3 and 4, peptidyl arginine deiminase 6, actin cytoplasmic 1, gamma-actin 1, pyruvate kinase, heat shock protein 90 and protein disulfide isomerase, among others. Major biological processes linked to the sheep embryo proteome were translation, protein transport and protein stabilization, and molecular functions, defined as ATP binding, oxygen carrier activity and oxygen binding. There were 42 enriched functional clusters according to the 2,147 genes (UniProt database). Ten selected clusters with potential association with embryo development included translation, structural constituent of ribosomes, ribosomes, nucleosomes, structural constituent of the cytoskeleton, microtubule-based process, translation initiation factor activity, regulation of translational initiation, cell body and nucleotide biosynthetic process. The most representative KEEG pathways were ribosome, oxidative phosphorylation, glutathione metabolism, gap junction, mineral absorption, DNA replication and cGMP-PKG signalling pathway. Analyses of functional clusters clearly showed differences associated with the proteome of preimplantation (D6) sheep embryos generated after in vitro fertilization in comparison with in vivo counterparts (Sanchez etal.,2021; https://doi.org/10.1111/rda.13897), confirming that the quality of in vitro derived blastocysts are unlike those produced in vivo. The present study portrays the first comprehensive overview of the proteome of preimplantational ovine embryos grown in vitro.

Proteomic profile of pre-implantational ovine embryos produced in vivo.

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.

Vitamin K2 improves developmental competency and cryo-tolerance of in vitro derived ovine blastocyst

Vitamin K2 (VK2), acts as an electron carrier in mitochondria and thereby effects reactive oxygen species (ROS) and ATP production. This study evaluates role of VK2 on in vitro developmental competency and cryo-survival of pre-implantation ovine embryos. Initially the optimal and beneficial concentration of VK2 on compaction and blastocyst formation rates was defined (0.1 μM). Subsequently, it was shown that 0.1 μM VK2, at blastocyst stage, reduces H2O2 production, increase the expression of mitochondrial related gene and improved embryos quality. We further assessed presence VK2 supplementation before and/or after vitrification of in vitro derived blastocysts. Our results reveal that presence of VK2 before and after vitrification improves rates of blastocysts re-expansion (88.19± 3.37% vs 73.68± 1.86%, P < 0.05) and hatching (49.55± 4.37% vs 32.7± 3.32%) compared to control group. These observation were consistent with reduction in H2O2 production and improved in expression of mitochondrial related genes. However, VK2 before or after vitrification, not only had no positive effect on these two parameters, but also significantly reduced these parameters. Therefore, in concordance with pervious report in bovine, we show that VK2 supplementation post genomic activation (Day 3–7) improved developmental competency of ovine in vitro derived embryos. We also showed that presence of VK2 after vitrification improves the cryo-survival of ovine embryos.

Expression pattern of OCT 4, NANOG and HSP 90 β gene in oocytes and various stages of in vitro produced sheep embryos

The study was conducted to assess the expression pattern of pluripotent genes (OCT 4 and NANOG) and stress response gene HSP 90 ß by quantitative real time polymerase chain reaction (qRT-PCR) in immature and mature oocytes and 2 cell, 4 cell, morula stages of in vitro fertilized (IVF) and parthenogenetically activated (PA) preimplantation ovine embryos to investigate the similarity between them in turn for the establishment of embryonic stem cells (ESCs) from parthenote embryos. The pluripotent related genes showed almost similar pattern of expression between IVF and PA embryos at all the developmental stages which indicated that PA embryos could be used as an alternate source for the production of embryonic stem cells. The stress response gene showed increased levels after in vitro maturation and non significant differences between IVF and PA embryos, thus, indicating no significant differences between the quantum of stress experienced by the 2 groups of embryos which would not affect the development of embryo quality and in turn affect the parthenote embryonic stem cells (PESCs).

Expression profile of FGF receptors in preimplantation ovine embryos and the effect of FGF2 and PD173074

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are increasingly recognized as important regulators of embryo development in mammals. This study investigated the importance of FGF signaling during in vitro development of ovine embryo. The mRNAs of four FGFR subtypes were detected throughout preimplantation development of in vitro fertilized (IVF) embryos, peaked in abundance at the morula stage, and decreased significantly at the blastocyst stage. To gain insight into the role of these mRNAs in embryo development, IVF embryos were cultured in the presence of FGF2 (100 or 500 ng/ml: beginning from days 1 or 4 to 7) or PD173074 (1 µM: beginning from days 1 to 7) as usual treatments for activation or inhibition of FGFRs, respectively. FGF2-supplementation did not affect the percentage of embryos that developed to the blastocyst, blastocyst cell count and the proportion of cells allocated in inner cell mass (ICM) and trophectoderm (TE) compared to control (p > 0.05). Also, increasing the dosage or duration of FGF2 treatment did not significantly alter blastocyst yield or differential cell count (p > 0.05). PD173074-mediated inhibition of FGFRs did not significantly affect blastocyst yield (p > 0.05). Assessment of expression profiles of lineage-associated markers revealed that FGF2 (500 ng/ml) supplementation: (i) significantly increased expression of putative hypoblast marker (GATA4), (ii) significantly decreased expression of putative epiblast (EPI) marker (NANOG) and (iii) did not change TE markers (CDX2 and IFNT) and pluripotency makers (OCT4, SOX2 and REX1). In summary, FGF2-mediated activation of FGFRs may promote a switch in transcriptional profile of ovine ICM from EPI- to hypoblast-associated gene expression.

130 EFFECT OF HYALURONIC ACID SUPPLEMENTATION ON OVINE EMBRYO DEVELOPMENT IN VITRO AND ON SURVIVAL AFTER VITRIFICATION

Studies have shown that supplementation with hyaluronic acid (HA), a glycosaminoglycan found in mammalian follicular, oviduct, and uterine fluids, improves in vitro development and post-thaw survival of bovine embryos. In this study, we examined the effect of HA supplementation on ovine embryo development and on survival after vitrification using the minimum volume cooling (MVC) cryotop method. Abattoir sourced ovine oocytes were in vitro matured and fertilized as per routine procedures (Walker et al. 1996 Biol. Reprod. 55, 703–708). In Experiment 1 (5 replicates), presumptive zygotes were randomly allocated to IVC medium supplemented with 0.8 mg mL–1 BSA, amino acids, and 0, 0.5, 1.0, 1.5 or 2.0 mg mL–1 HA. Cleavage rates were recorded and blastocyst development evaluated on Day 7 (Day 0 = day of IVF). In Experiment 2 (3 replicates), presumptive zygotes were placed in in vitro culture (IVC) medium with or without 1.0 mg mL–1 HA. Embryos were vitrified using the MVC cryotop method (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) on either Day 5 (morula–blastocyst stages), Day 6 (compact morula–hatching blastocyst stages), or Day 7 (blastocyst–hatching blastocyst stages). Vitrified embryos were thawed 7 days later and placed into IVC medium. Embryo survival (assessed by blastocoele re-expansion) and hatching rates were recorded on Day 8. Variables were assessed using procedure CATMOD in SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, the addition of HA did not affect cleavage or blastocyst formation rates but hatching rates were significantly (P &lt; 0.05) improved at concentrations of 0.5 to 1.5 mg mL–1 (Table 1). In Experiment 2, HA supplementation (1.0 mg mL–1) compared with control medium did not affect cleavage (96.8 and 97.1%, respectively) or blastocyst formation rates (68.6 and 70.2%, respectively). HA significantly (P &lt; 0.05) improved survival after thawing of embryos vitrified on Day 5 (100 v. 85.6%, n = 85 and 90). However no effect was observed when embryos were vitrified on either Day 6 (97.8 v. 97.8%, n = 91 and 92) or Day 7 (96.7 v. 97.9%, n = 92 and 94). Within day, HA supplementation did not affect hatching rate compared with control medium (Day 5, 54.1 v. 53.2%; Day 6, 62.9 v. 65.6%; Day 7, 71.9 v. 62.0%, respectively). These results demonstrate that HA supplementation of IVC medium significantly improves hatching rate of ovine embryos and we speculate that this improvement may correlate with comparable improvements in pregnancy rates after transfer. Hyaluronic acid binds to CD44, a glycoprotein expressed on the surface of preimplantation ovine embryos and shown to play a role on embryo development (Luz et al. 2012 Genet. Mol. Res. 11, 799–809). Hyaluronic acid plays a role in cell migration and we suggest that, in the early blastocyst, it affords an advantage to the trophectoderm cells. Table 1.Effect of hyaluronic acid (HA) supplementation in IVC medium on cleavage, blastocyst, and hatching rates

Different temporal gene expression patterns for ovine pre-implantation embryos produced by parthenogenesis or in vitro fertilization

Parthenogenetic activation of the mammalian oocyte constitutes an essential step to a number of oocyte- or embryo-related technologies. Mammalian parthenotes are useful tools for studying the roles of paternal and maternal genomes in early mammalian development and are considered potential candidates for an ethical source of embryonic stem cells. We investigated the in vitro developmental competence of pre-implantation ovine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) together with the expression of a panel of fourteen genes at different times of development. IVF and PA embryos showed similar developmental competence. No differences in gene expression were observed between PA and IVF two cell-stage embryos, while PA morulae showed a significantly higher expression of IGF2. At the blastocyst stage, parthenotes exhibited up-regulation of TP-1, CDC2, and IGF2 transcripts and significantly lower levels of AQP3, ATP1A1, H2A.Z, hsp90beta, and OCT4, while NANOG, BAX, CCNB1, CDH1, GAPDH, and IGF2R displayed similar expression patterns in the two groups. Our study indicates that oocyte parthenogenetic activation does not impair in vitro pre-implantation development to the blastocyst stage, but affects the gene expression status of the embryo after the activation of its own genome.

Targeted Suppression of Connexin 43 in Ovine Preimplantation Embryos by RNA Interference Using Long Double-stranded RNA

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.

TGFα and EGFR in ovine preimplantation embryos and effects on development

The present study aimed to assess location and relative amounts of transforming growth factor alpha (TGFα) and its receptor (EGFR) in ovine oocytes and preimplantation embryos by using immunohistochemical technique that was graded on a relative scale of 0–3, with 0 representing absence of staining, and 3 exhibiting prominent staining, and to evaluate the effects of TGFα/EGF on in vitro development of preimplantation embryos by adding different concentrations of EGF and TGFα to culture medium. The results showed that EGFR was abundant in cell plasma membranes in immature and mature oocytes, cumulus cells of immature cumulus-oocyte complexes (COC), fertilized oocytes and at different stages of embryo development. However, the relative amounts in inner cell mass (ICM) (1+) was less than that in trophectoderm (TE) cells (2+) at the blastocysts stage. The staining pattern for TGFα was a similar to EGFR. However, the staining for TGFα slightly increased in the fertilized oocytes (1–2+) as compared to immature and mature oocytes (1+). TGFα was mainly detected in the cytoplasm close to the membrane in both ICM and trophectoderm (TE) cells. The developmental rate of 8-cell stage embryos cultured with 5 ng/ml TGFα was increased as compared to other treatments ( P < 0.05). There was no significant difference in the rate of development of blastocysts cultured with 5 ng/ml TGFα, 20 ng/ml EGF, 20 ng/ml EGF + 5 ng/ml TGFα or the control treatment ( P > 0.05). In addition, there was no significant difference in the number of cells in blastocyst stage as compared with different treatments ( P > 0.05). However, TGFα alone enhanced cell survival rated ( P < 0.01) and reduced apoptosis. We concluded that TGFα can improve development of ovine preimplantation embryos at the 8-cell and blastocyst stages in vitro.

Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis

The major obstacle in the extensive analysis of the embryonic genome is the small number of cells typically obtained after the embryo biopsy. The object of the present study was to develop a simple approach that would allow the collection of a sufficient number of cells from a single embryo for use in further analyses. A micromanipulator was used to make a hole in the zona pellucida of 28 compacted morulae, 27 early blastocysts and 31 expanded blastocysts. After further culture, the trophoblastic cells, which herniated through this hole, were cut and cultured in vitro for different periods and used for embryo sexing. The results showed that biopsies can be taken successfully from 96.3% of early blastocysts, compared with 67.7% of expanded blastocysts and 71.4% of compacted morulae. The trophoblastic vesicles contained 20.8 +/- 6.7 cells (mean +/- SEM) and, when cultured, formed a confluent monolayer. The sex of cells cultured was assayed by PCR and the 12 lambs born after transfer of biopsied embryos confirmed its 100% accuracy. Moreover, no significant differences were found in the viability rates in vitro among blastocysts vitrified immediately after biopsy (77.8%), blastocysts biopsied and vitrified after 24 h culture (76.9%) and blastocysts vitrified without manipulation (88.5%). In experiments in vivo, the lambing rate of biopsied and vitrified blastocysts was significantly (P < 0.05) lower (40.0%) compared with vitrified control embryos (68.7%). This new approach to the biopsy of preimplantation embryos is a useful good model in the assisted reproductive technologies of domestic, wild and human species.

Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis

Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis

Vasoactive intestinal peptide influences hatching of ovine blastocysts.

Expanded blastocysts collected from superovulated Sarda ewes were divided at random into four groups for culture in a simple medium that does not support blastocyst hatching (CZB) or a complex medium that is permissive to hatching (TCM 199), with or without vasoactive intestinal peptide (VIP), a known embryo mitogenic peptide. Plasminogen activator (PA) secretion after 24 h of culture, and the number of cells, diameter of blastocysts and hatching rate after 48 h of culture were compared. The results showed an increase in hatching rate (78.6 v. 6.7%; P<0.01), diameter and number of cells (220.89 v. 210.44 microm, P<0.01 and 246 v. 232, P<0.01 respectively) and caseinolytic areas (1.33 v. 0.92 cm, P<0.01) of blastocysts cultured in TCM 199 compared with those cultured in CZB. Supplementation of the culture media with VIP increased these parameters in CZB (P<0.01) and partially in TCM 199. In particular, cell number, diameter and PA activity were significantly higher (P<0.01) after culture with VIP in both media. Immunoneutralization of exogenous VIP in culture with anti-VIP antibody caused a decrease in the hatching rate (P<0.01) of embryos cultured in medium with VIP, similar to the rate in unsupplemented CZB (P<0.01). These results suggest a receptor-mediated response. In immunohistochemical studies, VIP was shown to bind receptors in hatched blastocysts demonstrating the VIP-receptor interaction, and VIP receptors of approximately 150 kDa were revealed by electrophoretic studies. In conclusion, ovine preimplantation embryos exhibit VIP receptors, providing a basis for a receptor-mediated influence of VIP in the hatching of ovine blastocysts.

Induction of the heat shock response and translational thermotolerance in day 15 ovine trophectoderm

The objectives of this study were to determine the ability of trophectoderm from preimplantation ovine embryos to synthesize hsp70 in response to heat shock and to identify conditions which induce translational thermotolerance in this tissue. Day 15 embryos were collected, and proteins synthesized in 1.5-mm sections of trophectoderm were radioactively labeled with 35S-methionine. One-dimensional SDS-PAGE gels, two-dimensional gel electrophoresis and Western blots were utilized to characterize the heat shock response and to examine the induction of translational thermotolerance. Increased synthesis of the 70 kDa heat shock proteins and a protein with an approximate molecular weight of 15 to 20 kDa was observed with heat shock (≥ 42 °C). Total protein synthesis decreased (P < 0.05) with increased intensity of heat shock. At 45 °C, protein synthesis was suppressed with little or no synthesis of all proteins including hsp70. Recovery of protein synthesis following a severe heat shock (45 °C for 20 min) occurred faster (P < 0.05) in trophectoderm pretreated with a mild heat shock (42 °C for 30 min) than trophectoderm not pretreated with mild heat. In summary, trophoblastic tissue obtained from ovine embryos exhibit the characteristic “heatshock” response similar to that described for other mammalian systems. In addition, a sublethal heat shock induced the ability of the tissue to resume protein synthesis following severe heat stress. Since maintaining protein synthesis is crucial to embryonic survival, manipulation of the heat-shock response may provide a method to enhance embryonic survival.

Ultrastructural localization of lectin receptors in the preimplantation ovine embryo

Preimplantation development of mammalia is characterized by cell surface changes functioning in intercellular communication and adhesion. The glycoconjugate role in cellular interactions has been analysed for several groups but not in sheep embryos. The binding patterns of eleven lectins during sheep preimplantation development were investigated and the role of glycoconjugates in early development was discussed. Ultrathin sections from preimplantation ovine embryos (3-7 days) were incubated with different colloidal gold conjugated lectins and the frequency of gold particles on the cell membrane, some organelles, and the zona pellucida was evaluated. We observed a higher staining of WGA, DBA, and SBA lectins in the intercellular contact zone with respect to the free cell surface of blastomeres during cleavage. This indicates that the N-acetyl galactosamine and N-acetyl glucosamine residues may be involved in sheep morula compaction. In contrast, the trophoblast cell displays an increase of staining of some lectins previously identified during cleavage (LcH, WGA, SBA, MPA, and PNA) on the free membrane, and a lack of sugar residues in the intercellular surface. This polarization of the trophoblast cell surface is not observed in the inner cell mass and could provide a mechanism for differentiation within the blastocyst. Intracytoplasmic vesicles show a cytochemical identity with lysosomes in the blastocyst (abundant GlcNAc and Man/Glc residues) that may reflect a functional relationship between both organelles in an intracellular cycle. The zona pellucida presents abundant GalNAc, GlcNAc, and Gal residues during preimplantation ovine development.

Noninvasive measurement of pyruvate uptake by ovine preimplantation embryos and unfertilized ova

A noninvasive, fluorometric assay was used to measure pyruvate uptake by ovine unfertilized ova and preimplantation embryos up to the expanded blastocyst stage. There was a significant (P<0.02) increase in pyruvate uptake between unfertilized ova and one-cell embryos, after which uptake levels declined. Another significant increase (P<0.02) was seen at the early morula stage, with uptake levels remaining high through the expanded blastocyst stage. The pattern of pyruvate uptake observed was markedly different from that which has been reported in mice. These data suggest that the metabolic events of preimplantation embryonic development may differ substantially between the mouse and sheep.

Detection of histocompatibility-Y antigen: identification of sex of pre-implantation ovine embryos

Indirect immunofluorescence was used to detect the presence of a male-specific protein (H-Y antigen) on pre-implantation ovine embryos. Eight-cell to blastocyst stage embryos were surgically collected from superovulated ewes, classified as fluorescent (H-Y positive) or non-fluorescent (H-Y negative) and either transferred to recipient ewes or karyotyped to confirm embryonic sex. H-Y antigen was detected on eight-cell through blastocyst stage embryos. Overall, 88% ( 50 57 ) of the embryos (eight-cell to early blastocyst stage) classified as H-Y positive or H-Y negative were male and female, respectively. Survival after transfer of embryos subjected to the H-Y antigen assay was high (63%), which supports the use of this procedure in conjunction with embryo transfer in sheep to produce pregnancies in which sex of the fetus is known.