Mohamed Ashry

Characterization of the molecular factors regulating early embryonic development and their functional mechanisms is critical for understanding the causes of early pregnancy loss in monotocous species (cattle, human). We previously characterized a stage specific functional role of follistatin, a TGF-beta superfamily binding protein, in promoting early embryonic development in cattle. The mechanism by which follistatin mediates this embryotropic effect is not precisely known as follistatin actions in cattle embryos are independent of its classically known activin inhibition activity. Apart from activin, follistatin is known to bind and modulate the activity of the bone morphogenetic proteins (BMPs), which signal through SMAD1/5 pathway and regulate several aspects of early embryogenesis in other mammalian species. Present study was designed to characterize the activity and functional requirement of BMP signaling during bovine early embryonic development and to investigate if follistatin involves BMP signaling for its stage specific embryotropic actions. Immunostaining and western blot analysis demonstrated that SMAD1/5 signaling is activated after embryonic genome activation in bovine embryos. However, days 1-3 follistatin treatment reduced the abundance of phosphorylated SMAD1/5 in cultured embryos. Inhibition of active SMAD1/5 signaling (8-16 cell to blastocyst) using pharmacological inhibitors and/or lentiviral-mediated inhibitory SMAD6 overexpression showed that SMAD1/5 signaling is required for blastocyst production, first cell lineage determination as well as mRNA and protein regulation of TE (CDX2) cell markers. SMAD1/5 signaling was also found to be essential for embryotropic actions of follistatin during days 4-7 but not days 1-3 of embryo development suggesting a role for follistatin in regulation of SMAD1/5 signaling in bovine embryos.

Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-β regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72 h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.

BACKGROUND: TGF-β signaling pathways regulate several crucial processes in female reproduction. AKT is a non-SMAD signaling pathway regulated by TGF-β ligands essential for oocyte maturation and early embryonic development in the mouse, but its regulatory role in bovine early embryonic development is not well established. Previously, we demonstrated a stimulatory role for follistatin (a binding protein for specific members of TGF-β superfamily) in early bovine embryonic development. The objectives of the present studies were to determine the functional role of AKT signaling in bovine early embryonic development and embryotrophic actions of follistatin.

METHODS: We used AKT inhibitors III and IV as pharmacological inhibitors of AKT signaling pathway during the first 72 h of in vitro embryo culture. Effects of AKT inhibition on early embryonic development and AKT phosphorylation were investigated in the presence or absence of exogenous follistatin.

RESULTS: Pharmacological inhibition of AKT signaling resulted in a significant reduction in early embryo cleavage, and development to the 8- to 16-cell and blastocyst stages (d7). Treatment with exogenous follistatin increased AKT phosphorylation and rescued the inhibitory effect of AKT inhibitors III and IV on AKT phosphorylation and early embryonic development.

CONCLUSIONS: Collectively, results suggest a potential requirement of AKT for bovine early embryonic development, and suggest a potential role for follistatin in regulation of AKT signaling in early bovine embryos.

Zinc finger (ZNF) transcription factors interact with DNA through zinc finger motifs and play important roles in a variety of cellular functions including cell growth, proliferation, development, apoptosis, and intracellular signal transduction. One-third of ZNF proteins in metazoans contain a highly conserved N-terminal motif known as the Krüppel-associated box (KRAB) domain, which acts as a potent, DNA-binding dependent transcriptional repression module. Analysis of RNA-Seq data generated from a bovine oocyte cDNA library identified a novel transcript, which encodes a KRAB-containing ZNF transcription factor (named ZNFO). Characterization of ZNFO mRNA expression revealed that it is exclusively expressed in bovine oocytes and early embryos. A GFP reporter assay demonstrated that ZNFO protein localizes specifically to the nucleus, supporting its role in transcriptional regulation. To test the role of ZNFO in early embryonic development, zygotes were generated by in vitro maturation and fertilization of oocytes, and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was significantly reduced relative to the uninjected and negative control siRNA-injected embryos. Further, interaction of ZNFO with the highly conserved co-factor, KRAB-associated protein-1 (KAP1), was demonstrated, and evidence supporting transcriptional repression by ZNFO was demonstrated using a GAL4-luciferase reporter system. Results of described studies demonstrate that ZNFO is a maternally-derived oocyte-specific nuclear factor required for early embryonic development in cattle, presumably functioning by repressing transcription.

The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus-oocyte complexes (COCs) from BCB+, BCB- and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB- and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.

Brilliant cresyl blue (BCB) is a super vital stain that has been used to select competent oocytes in different species. The objectives of the first part of the present studies were to determine mRNA abundance for select TGFβ superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels and transcript abundance for other oocyte (JY1) and cumulus cell (CTSB, CTSK, CTSS and CTSZ) markers of oocyte quality in bovine oocytes and or adjacent cumulus cells classified based on developmental potential using BCB staining. The ability of exogenous FST, JY1, or cathepsin inhibitor treatment to rescue development of embryos derived from poor quality oocytes selected based on BCB staining was also determined. Cumulus oocyte complexes (COCs) from abattoir derived ovaries were subjected to BCB staining and GV stage oocytes and cumulus cells harvested from control, BCB+ and BCB- (poor oocyte quality) groups for real time PCR or Western blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization and embryo culture in presence or absence of above described treatments. Levels of FST, JY1, BMP15 and SMAD1, 2, 3 and 5 transcripts were higher in BCB+ oocytes whereas abundance of CTSB, CTSK, CTSS and CTSZ mRNAs was higher in cumulus cells surrounding poor quality BCB- oocytes. Western blot analysis revealed SMAD1/5 and SMAD2/3 phosphorylation were higher in BCB+ than BCB- oocytes. Embryo culture studies demonstrated that follistatin and cathepsin inhibitor treatment but not JY-1 treatment can rescue developmental competence of BCB- oocytes. Results provide further understanding of molecular indices of oocyte competence. The focus of the second part of the present studies was d to elucidate the regulatory role of protein kinase B “AKT” in oocytes and embryo competence and potential link to the embryotrophic actions of FST. The objectives of these studies were to determine the relationship between AKT transcript abundance/signaling activity and oocyte competence determined by BCB staining, and characterize the temporal changes in AKT mRNA during oocyte maturation and early embryogenesis in vitro. Effects of AKT inhibition on early embryonic progression and effects of follistatin supplementation on developmental capacity of AKT inhibitor treated embryos and signaling activity of AKT and its downstream targets were also analyzed. In vitro embryo production model was utilized to study the effect of AKT inhibition and FST supplementation on early embryos, qRT-PCR and Western blot were used for analysis of mRNA transcript abundance and signaling activity of investigated pathways respectively. Both AKT mRNA and phosphorylation level were higher in BCB+ than BCB- oocytes. Abundance of mRNA for AKT was increased in pronuclear through 8-cell stage embryos relative to GV stage oocytes, then decreased at 16-cell stage and further decreased in morula and blastocyst stage embryos. AKT inhibition during the initial 72 h of embryo culture blocks early cleavage, reduces total cleavage, 8-16 cell stage and blastocyst formation rate. FST supplementation partly rescues the effects of AKT inhibition but didn’t affect the phosphorylation level of AKT despite the significant increase in p-AKT at 1 and 10 h after FST supplementation. Results suggest a positive relationship between AKT transcript abundance/ signaling activity and oocyte competence determined by BCB staining. Results also demonstrate a temporal regulation of AKT mRNA abundance during early embryogenesis and indicate that embryotrophic actions of exogenous FST may be mediated, at least in part, by modulation of AKT signaling pathway. Further studies are required to elucidate the functional role of AKT and mechanism of action of FST in regulation of early embryonic development in bovine.

Dogs have become an important household pet in Egypt beside their valuable part in the community, police and army forces and rescue organizations. The current study aimed at utilizing some important diagnostic techniques such as vaginal cytology and ultrasonography evaluation and improvement of canine reproduction.The current study was conducted on 45 out-patient bitches admitted to the Theriogenology Clinic from November 2008 to June 2009 for pregnancy diagnosis. These bitches were assigned into 2 groups, pregnant (28) and non pregnant (17). Based on vaginal cytology and ultrasonography, the non pregnant group bitches were evaluated and assigned into 7 bitches in anestrus,3 bitches in proestrus, 3 in estrus and 4 bitches in diestrus.Ultrasound examinations of pregnant group revealed that pregnancy was first detected 18 – 24 days post mating, 24 – 25 days from LH surge.Characterization of normal prenatal development was described and used to estimate fetal age. Fetal viability via heart beats was first assessed at 25 – 27days and via fetal movement at 34 – 36 days. Estimation of litter size was accurately done between 25 and 35 days. Parturition date was predicted and used to apply elective cesarean section with delivery of viable healthy puppies.In conclusion, we inferred that vaginal cytology is a simple, rapid and inexpensive test to confirm estrus and help an owner to determine the proper time to breed a bitch. Ultrasonography is a very valuable diagnostic tool to diagnose early pregnancy, estimate litter size, monitor fetal viability and predict parturition date; in addition, to differentiate between pregnancy and pyometra and other abnormalities.