Samantha M. Calcatera

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality.

Effects of fescue toxicosis on bull growth, semen characteristics, and breeding soundness evaluation.

Tall fescue possesses heat, drought, and pest resistance conferred to the plant by its mutualistic relationship with the ergot alkaloid producing fungal endophyte, Neotyphodium coenophialum. The objective of this study was to evaluate the impact of ergot alkaloid consumption on growth, scrotal circumference (SC), and semen quality. The SC measurement and percentage of motile and normal sperm were used to determine if a bull passed the breeding soundness exam (BSE) requirements. Bulls (n = 14) between 13 and 16 mo of age exhibiting ≥32 cm SC and having passed a BSE were assigned to 1 of 2 dietary treatments accounting for BCS and BW. Bulls were fed the treatment diet containing toxic tall fescue seed (E+; 0.8 μg of ergovaline and ergovalanine/g DM) or the control diet containing endophyte-free nontoxic tall fescue seed (E-) for 126 d. Blood samples were collected and BSE and BCS accessed at the start of the test (d 0) and every 21 d to the end of test (d 126). Weights were obtained on d 0 and d 126. Serum prolactin (PRL) concentrations were affected by treatment × day interactions (P = 0.04) verifying the effectiveness of the E+ diet. Bulls consuming the E+ diet exhibited declining PRL concentrations from 250 ± 52.1 ng/mL on d 0 to 30.6 ± 46.9 ng/mL by d 126 whereas bulls receiving the E- ration maintained serum PRL concentrations greater than or equal to 226.7 ± 50.4 ng/mL across the 126-d study. Body condition score (P = 0.4) and BW (P = 0.4) were not different between treatments. No difference due to treatment was observed for the percentage of bulls passing a standard BSE exam (P = 0.6) and no treatment effect was observed for any semen characteristic measured by computer-assisted semen analysis (CASA; P ≥ 0.2). The SC was negatively affected by treatment × day interaction (P = 0.04) with E- bulls exhibiting a larger SC at d 126 compared with E+ bulls of 36.7 ± 0.8 versus 34.3 ± 0.8 cm, respectively. Within treatment, E+ bulls exhibited a decrease in SC (P = 0.0001) with a d 0 SC of 37.3 ± 0.8 cm and dropping to 34.3 ± 0.8 by d 126. Theoretically, reduced SC would negatively impact semen quality, but this was not observed. However, CASA and BSE evaluation data are consistent with recent reports indicating that bulls grazing E+ tall fescue exhibited only subtle, if any, differences on semen characteristics.

The bull sperm microRNAome and the effect of fescue toxicosis on sperm microRNA expression.

Tall fescue [Schedonorus phoenix (Scop.) Holub] accounts for nearly 16 million hectares of pasture in the Southeastern and Mid-Atlantic U.S. due to its heat, drought, and pest resistance, conferred to the plant by its symbiotic relationship with the endophyte Neotyphodium coenophialum. The endophyte produces ergot alkaloids that have negative effects on the growth and reproduction of animals, resulting in the syndrome known as fescue toxicosis. The objectives of our study were to identify microRNA (miRNA) present in bovine sperm and to evaluate the effects of fescue toxicosis on sperm miRNA expression. Angus bulls were assigned to treatments of either toxic or non-toxic fescue seed diets. Semen was collected and subjected to RNA isolation. Three samples from each treatment group were chosen and pooled for deep sequencing. To compare miRNA expression between treatment groups, a microarray was designed and conducted. For each of the top ten expressed miRNA, target prediction analysis was conducted using TargetScan. Gene ontology enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery. Sequencing results elucidated the presence of 1,582 unique small RNA present in sperm. Of those sequences, 382 were known Bos taurus miRNA, 22 were known but novel to Bos taurus, and 816 were predicted candidate miRNA that did not map to any currently reported miRNA. Of the sequences chosen for microarray, twenty-two showed significant differential expression between treatment groups. Gene pathways of interest included: regulation of transcription, embryonic development (including blastocyst formation), Wnt and Hedgehog signaling, oocyte meiosis, and kinase and phosphatase activity. MicroRNA present in mature sperm appears to not only be left over from spermatogenic processes, but may actually serve important regulatory roles in fertilization and early developmental processes. Further, our results indicate the possibility that environmental changes may impact the expression of specific miRNA.

Expression of microRNA in male reproductive tissues and their role in male fertility

MicroRNA (miRNA) are small non-coding RNA, approximately 22 nucleotides in length, that regulate gene expression through their ability to bind to mRNA. The role of miRNA in cellular and tissue development is well documented and their importance in male reproductive tissue development is actively being evaluated. They are present in spermatogonia, Sertoli and Leydig cells within the testis and are present in mature spermatozoa, indicating roles in normal testicular development, function and spermatogenesis. Their presence in spermatozoa has led to postulations about the roles of male miRNA during early embryonic development after fertilisation, including chromatin restructuring and possible epigenetic effects on embryo development. MiRNAs are also present in body fluids, such as blood serum, milk, ovarian follicular fluid and seminal fluid. Circulating miRNAs are stable, and aberrant expression of cellular or extracellular miRNA has been associated with multiple pathophysiological conditions, the most studied being numerous forms of cancer. Considering that miRNAs are present in spermatozoa and in seminal fluid, their stability and the relatively non-invasive procedures required to obtain these samples make miRNAs excellent candidates for use as biomarkers of male reproduction and fertility. Biomarkers, such as miRNAs, identifying fertile males would be of financial interest to the animal production industry.

Immunohistochemical detection of Argonaute 2 in bovine and porcine tissues

Abstract Argonaute 2 (Ago2) is an RNA binding protein that possesses RNase III activity and is a key component of the microRNA pathway in which it cleaves double-stranded RNA. Ago2 is required for essential cellular processes and plays a role in dictating translation of proteins. Commercial antibodies have been utilized for the detection of human and mouse Ago2 using immunohistochemistry (IHC) and western blotting; however, data describing detection in additional mammalian species are currently lacking. Therefore, we evaluated the use of a commercial polyclonal antibody in detecting Ago2 in bovine and porcine tissues. Tissue samples were collected at slaughter and either flash-frozen for western blotting or fixed in 10% neutral buffered formalin and embedded in paraffin for IHC. When bovine and porcine tissue extracts were analyzed by western blotting, anti-Ago2 detected a predominant protein band of ∼108 kDa which closely corresponds to the predicted molecular weight for Ago2; however, multiple bands of lesser intensity and molecular weight were also detected. Using the same antibody, fluorescent IHC on paraffin sections resulted in positive staining for both bovine and porcine tissues including kidney, liver, pancreas, and spleen. Staining for Ago2 was localized in the cytoplasm and appeared to be cell type-specific, with higher signal intensity in endothelial cells, leukocytes, and endocrine cells.