Na/K-ATPase beta1 subunit expression is required for blastocyst formation and normal assembly of trophectoderm tight junction-associated proteins.

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase alpha and beta isoforms during mouse preimplantation development, only the alpha1 and beta1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/K-ATPase beta1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealthtrade mark Na/K-ATPase beta1 subunit oligos or nonspecific Stealthtrade mark siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase beta1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase beta1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase beta1 subunit mRNAs and protein were not detectable in beta1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between beta1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase alpha1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase beta1 subunit siRNA-microinjected embryos. Our results demonstrate that the beta1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development

Application of imaging and spectroscopy techniques for grading of bovine embryos - a review

Although embryo transfers have grown considerably in the cattle industry, the selection of embryos required for successful pregnancies remains a challenging task. Visual inspection of 7th-day embryos using a stereomicroscope, followed by classification based on morphological features is the most commonly practiced procedure. However, there are inaccuracies and inconsistencies in the manual grading of bovine embryos. The objective of this review was to evaluate the potential of imaging and spectroscopic techniques in the selection of bovine embryos. Digital analysis of microscopic images through extracting visual features in the embryo region, and classification using machine learning methods have yielded about 88–96% success in pregnancies. The Raman spectral pattern provides valuable information regarding developmental stages and quality of the embryo. The Raman spectroscopy approach has also been successfully used to determine various parameters of bovine oocytes. Besides, Fourier Transform Infrared (FTIR) spectroscopy has the ability to assess embryo quality through analyzing embryo composition, including nucleic acid and amides present. Hyperspectral Imaging has also been used to characterize metabolite production during embryo growth. Although the time-lapse imaging approach is beneficial for morphokinetics evaluation of embryo development, optimized protocols are required for successful implementation in bovine embryo transfers. Most imaging and spectroscopic findings are still only at an experimental stage. Further research is warranted to improve the repeatability and practicality to implement in commercial facilities

The p66\u3csup\u3eShc\u3c/sup\u3e adaptor protein controls oxidative stress response in early bovine embryos

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos. © 2014 Betts et al

Mitogen-activated protein kinase (MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase (ERK) pathways.

Blastocyst formation, as a critical period during development, is an effective indicator of embryonic health and reproductive efficiency. Out of a number of mechanisms underlying blastocyst formation, highly conserved mitogen-activated protein kinase (MAPK) signaling has emerged as a major mechanism involved in regulating murine preimplantation embryo development. The objective of our study was to ascertain the role of MAPK signaling in regulating bovine development to the blastocyst stage. Using reverse transcriptase PCR and immunohistochemical staining procedures we have demonstrated that mRNA transcripts and polypeptides encoding p38 MAPK pathway constituents are detectable in preimplantation bovine embryos from the one-cell to the blastocyst stage. Further, the effects on bovine embryo development following inhibition of p38 alpha/beta and extracellular signal-regulated kinase (ERK) signaling by treatment with SB220025 and U0126, respectively, were investigated. Eight-cell bovine embryos (50 per group; three replicates) were placed into treatments consisting of synthetic oviductal fluid (SOF) medium: SOF + SB202474 (inactive analogue), SOF + SB220025, SOF + U0124 (inactive analogue), SOF + U0126, and SOF + SB220025 + U0126. Inhibition of p38 MAPK or ERK signaling individually did not affect development to the blastocyst stage. However, when both pathways were blocked simultaneously there was a significant reduction (P \u3c 0.05) in blastocyst formation, cell number and immunofluorescence of phosphorylated downstream pathway constituents. We have determined that, in variance to what was observed during murine preimplantation development, bovine early embryos progress at normal frequencies to the blastocyst stage in the presence of p38 MAPK inhibitors

Na+/K+ -ATPase regulates tight junction formation and function during mouse preimplantation development.

Research applied to the early embryo is required to effectively treat human infertility and to understand the primary mechanisms controlling development to the blastocyst stage. The present study investigated whether the Na(+)/K(+)-ATPase regulates tight junction formation and function during blastocyst formation. To investigate this hypothesis, three experimental series were conducted. The first experiments defined the optimal dose and treatment time intervals for ouabain (a potent and specific inhibitor of the Na(+)/K(+)-ATPase) treatment. The results demonstrated that mouse embryos maintained a normal development to the blastocyst stage following a 6-h ouabain treatment. The second experiments investigated the effects of ouabain treatment on the distribution of ZO-1 and occludin (tight junction associated proteins). Ouabain treatment (up to 6 h) or culture in K(+)-free medium (up to 6 h) resulted in the appearance of a discontinuous ZO-1 protein distribution and a loss of occludin immunofluorescence. The third set of experiments examined the influence of ouabain treatment on tight junction function. Ouabain treatment or culture in K(+)-free medium affected tight junction permeability as indicated by an increase in the proportion of treated embryos accumulating both 4 kDa and 40 kDa fluorescein isothiocyanate (FITC)-dextran into their blastocyst cavities. The results indicate that the Na(+)/K(+)-ATPase is a potent regulator of tight junction formation and function during mouse preimplantation development

The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro

<p>Abstract</p> <p>Background</p> <p>Excessive developmental failure occurs during the first week of <it>in vitro </it>embryo development due to elevated levels of cell death and arrest. We hypothesize that permanently arrested embryos enter a stress-induced "senescence-like" state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66Shc. The aim of this study was to selectively diminish p66Shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by RNA-mediated interference to study the effects of p66Shc knockdown on <it>in vitro </it>fertilized bovine embryos.</p> <p>Results</p> <p>Approximately 12,000–24,000 short hairpin (sh)RNAi molecules specific for p66Shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. Experiments were comprised of a control group undergoing IVF alone and two groups microinjected with and without p66Shc shRNAi molecules prior to IVF. The amount of p66Shc mRNA quantified by Real Time PCR was significantly (P < 0.001) lowered upon p66Shc shRNAi microinjection. This reduction was selective for p66Shc mRNA, as both histone H2a and p53 mRNA levels were not altered. The relative signal strength of p66Shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66Shc shRNAi microinjected groups compared to controls (P < 0.05). A significant decrease (P < 0.001) in the incidence of arrested embryos upon p66Shc shRNAi microinjection was detected compared to IVF and microinjected controls along with significant reductions (P < 0.001) in both cleavage divisions and blastocyst development. No significant differences in p66Shc mRNA levels (P = 0.314) were observed among the three groups at the blastocyst stage.</p> <p>Conclusion</p> <p>These results show that p66Shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos.</p

Characterization of cadherins, adamts, steroid receptors, during blastocyst formation in vitro of adamts in the bovine ovary

In bovine, early embryonic mortality (EEM) results in low reproductive efficiency, the major cause for which is believed to be impaired development of the embryo, but mechanisms involved therein remain poorly understood. A lack of progesterone support from the corpus luteum (CL) also contributes to EEM. Recent studies demonstrate that regulated expression of distinct cadherins that mediate Ca²⁺ - dependent cell-cell adhesion and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) subtypes involved in cell-matrix interactions, and steroid receptors mediate important cellular events underlying the formation and organization of tissues, however, their role in preimplantation embryogenesis is poorly characterized. Understanding genes involved in the blastocyst and CL formation provides interesting possibilities in improving embryo viability. To address these issues, investigations into the expression of cadherins, ADAMTS, estrogen and progesterone receptor isoforms during the preimplantation embryogenesis in bovine embryos at timed stages of development has been undertaken, using reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemistry strategies. In addition, expression of ADAMTS subtypes in ovarian follicles and CL has also been characterized. These studies indicate that multiple cadherins, both type 1 (E-, N - and P-Cad) and type 2 (cad-8, -10 and -11), with their specific localizations, at least in part, provide the molecular basis for the formation and organization of the inner cell mass and the trophectoderm. The differential mRNA and protein expression of steroid receptor isoforms (ERα, ERβ, PRA and PRB) suggests distinct role(s) during early embryogenesis and provides evidence for direct effect of steroids on the embryo. The regulated but complex expression of ADAMTS subtypes both in the embryo (ADAMTS-1, -4, -5, -6, -8, -9, -10, but not ADAMTS-2, -3, -7 or -12) and the ovary (ADAMTS-1, -2, -3, -4, -5, -7, -8, -9, but not ADAMTS-6, -10 or -12) suggests that these proteinases could be involved in cell-matrix interactions and contributing towards important cellular events like cell migration, differentiation and remodeling of the extracellular matrix. Though, the biological significance of these molecules remains unclear, these studies demonstrate that novel cellular molecules involved in cell-cell, cell-matrix and steroid interactions could be playing a key role during developmental processes like embryogenesis and/or CL formation.Land and Food Systems, Faculty ofGraduat

A Comparative Analysis of Hippo Signaling Pathway Components during Murine and Bovine Early Mammalian Embryogenesis

The time required for successful blastocyst formation varies among multiple species. The formation of a blastocyst is governed by numerous molecular cell signaling pathways, such as the Hippo signaling pathway. The Hippo signaling pathway is initiated by increased cell–cell contact and via apical polarity proteins (AMOT, PARD6, and NF2) during the period of preimplantation embryogenesis. Cell–cell contact and cell polarity activate (phosphorylates) the core cascade components of the pathway (mammalian sterile twenty like 1 and 2 (MST1/2) and large tumor suppressor 1 and 2 (LATS1/2)), which in turn phosphorylate the downstream effectors of the pathway (YAP1/TAZ). The Hippo pathway remains inactive with YAP1 (Yes Associated protein 1) present inside the nucleus in the trophectoderm (TE) cells (polar blastomeres) of the mouse blastocyst. In the inner cell mass (ICM) cells (apolar blastomeres), the pathway is activated with p-YAP1 present in the cytoplasm. On the contrary, during bovine embryogenesis, p-YAP1 is exclusively present in the nucleus in both TE and ICM cells. Contrary to mouse embryos, transcription co activator with PDZ-binding motif (TAZ) (also known as WWTR1) is also predominantly present in the cytoplasm in all the blastomeres during bovine embryogenesis. This review outlines the major differences in the localization and function of Hippo signaling pathway components of murine and bovine preimplantation embryos, suggesting significant differences in the regulation of this pathway in between the two species. The variance observed in the Hippo signaling pathway between murine and bovine embryos confirms that both of these early embryonic models are quite distinct. Moreover, based on the similarity of the Hippo signaling pathway between bovine and human early embryo development, bovine embryos could be an alternate model for understanding the regulation of the Hippo signaling pathway in human embryos

Senescence or apoptosis? The choice bovine fibroblasts make in the presence of increasing concentrations of extracellular H2O2

Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been linked to the regulation of specific cellular functions, including apoptosis. In response to specific signals, mitochondrial H2O2 can trigger mitochondrial swelling and apoptosis; however, as a protective measure, cells can also enter a state of senescence. The purpose of this study was to determine the effects of extracellular H2O2 on cellular senescence induction via the mitochondrial ROS-signaling pathway. Bovine fibroblasts were synchronized at the G1/S phase using a thymidine-nocodazole mitotic block and treated for two hours with H2O2 concentrations: 25μM, 50μM, 100μM, 150μM and 200μM. Post H2O2 treatment, the number of floating dead cells was quantified using a hemocytometer. Cells sticking to the bottom of the culture dish were then washed with fresh media and cultured for 24 hours at 39oC at 5% CO2 under humidified conditions and assayed for senescent cell quantification using SA--galactosidase staining assay. Preliminary data indicates that the number of dead cells increased with increasing H2O2 dosage, with the maximum number of dead cells at 200m in both cell lines. Along with the increase in number of dead cells, the number of senescent cells decreased up to 50M, and no senescent cells were detected at 50m concentrations. This indicates that cells are sensitive to rising levels of H2O2 and make a choice to enter the senescent or apoptotic pathway based on the oxidative insult they face. Future studies would focus on the molecular mechanism of the mitochondrial-ROS signaling pathway

The effect of p66Shc knockdown on H₂O₂-induced permanent embryo arrest^*.

¥<p>n = 100/replicate, 3 replicates.</p>*<p>Superscript letters denote significant differences (P<0.05) within groups.</p