Rajput, S. K., C. Yang, M. Ashry, J. K. Folger, J. G. Knott, and G. W. Smith, "Role of bone morphogenetic protein signaling in bovine early embryonic development and stage specific embryotropic actions of follistatin†.", Biology of reproduction, vol. 102, issue 4, pp. 795-805, 2020. Abstract

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.

Fathi, M., M. Ashry, K. M. Ali, A. Hassan, and A. F. Elkarmoty, "Clinico-Pathological Evaluation and Treatment Outcomes of Canine Transmissible Venereal Tumor Using Three Different Protocols", Pakistan Veterinary journal, vol. 38, issue 2, pp. 204-208, 2018.
Ashry, M., K. B. Lee, J. K. Folger, S. K. Rajput, and G. W. Smith, "Follistatin supplementation during in vitro embryo culture improves developmental competence of bovine embryos produced using sex-sorted semen.", Reproductive biology, vol. 18, issue 3, pp. 267-273, 2018 Sep. Abstract

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.

Waheeb, R., M. Ashry, A. B. A. Ali, and G. Amrawi, "Effects of oral administration of Gonadotrophin Stimulant (Theriogon®) on sexual behavior and semen characteristics in bulls", Asian Journal of Animal and Veterinary Advances, 2018.
Folger, J. K., M. Ashry, S. K. Rajput, and G. W. Smith, "FSH stimulated steroidogenesis involves both canonical and non-canonical WNT signaling in bovine granulosa cells", 51th Annual Meeting of Society for the Study of Reproduction, New Orleans, Louisiana, USA, 10-13 July, 2018.
Ashry, M., K. B. Lee, J. K. Folger, S. K. Rajput, and G. W. Smith, "Follistatin Supplementation During In Vitro Embryo Culture Improves Developmental Competence of Bovine Embryos Produced Using Sex-sorted semen", 51th Annual Meeting of Society for the Study of Reproduction, New Orleans, Louisiana, USA, 10-13 July, 2018.
Ashry, M., S. K. Rajput, J. K. Folger, J. G. Knott, N. A. Hemeida, O. M. Kandil, R. S. Ragab, and G. W. Smith, "Functional role of AKT signaling in bovine early embryonic development: potential link to embryotrophic actions of follistatin.", Reproductive biology and endocrinology : RB&E, vol. 16, issue 1, pp. 1, 2018 Jan 08. Abstract

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.

Ashry, M., S. Rajput, J. Folger, and G. Smith, "Potential Role for WNT Signaling in FSH Regulated Ovarian Steroidogenesis in cattle", MSU Reproductive and Developmental Sciences Program, First Annual Research Day, East Lansing, MI, USA, November 16, 2017.
Fathi, M., M. Ashry, A. Salama, and M. R. Badr, "Developmental competence of Dromedary camel (Camelus dromedarius) oocytes selected using brilliant cresyl blue staining.", Zygote , pp. 1-8, 2017 Jul 11. Abstract

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.

Hand, J. M., K. Zhang, L. Wang, Prasanthi P. Koganti, K. Mastrantoni, S. K. Rajput, M. Ashry, G. W. Smith, and J. Yao, "Discovery of a novel oocyte-specific Krüppel-associated box domain-containing zinc finger protein required for early embryogenesis in cattle.", Mechanisms of development, vol. 144, pp. 103-112, 2017 Mar 02. Abstract

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.