Cai-Xia Yang

Small RNA Profile of the Cumulus-Oocyte Complex and Early Embryos in the Pig

ABSTRACT Small RNA represent several unique noncoding RNA classes that have important function in the development of germ cells and early embryonic development. Deep sequencing was performed on small RNA from cumulus cells (recovered from germinal vesicle [GV] and metaphase II-arrested [MII] oocytes), GV and MII oocytes, in vitro fertilization-derived embryos at 60 h postfertilization (4- to 8-cell stage), and Day 6 blastocysts. Additionally, a heterologous miRNA microarray method was also used to identify miRNA expressed in the oocyte during in vitro maturation. Similar to the results of expression analysis of other species, these data demonstrate dynamic expression regulation of multiple classes of noncoding RNA during oocyte maturation and development to the blastocyst stage. Mapping small RNA to the pig genome indicates dynamic distribution of small RNA organization across the genome. Additionally, a cluster of miRNA and Piwi-interacting RNA (piRNA) was discovered on chromosome 6. Many of the small RNA mapped to annotated repetitive elements in the pig genome, of which the SINE/tRNA-Glu and LINE/L1 elements represented a large proportion. Two piRNA (piR84651 and piR16993) and seven miRNA (MIR574, MIR24, LET7E, MIR23B, MIR30D, MIR320, and MIR30C) were further characterized using quantitative RT-PCR. Secretory carrier membrane protein 4 (SCAMP4) was predicted to be subject to posttranscriptional gene regulation mediated by small RNA, by annotating small RNA reads mapped to exonic regions in the pig genome. Consistent with the prediction results, SCAMP4 was further confirmed to be differentially expressed at both transcriptional and translational levels. These data establish a small RNA expression profile of the pig cumulus-oocyte complex and early embryos and demonstrate their potential capacity to be utilized for predictions of functional posttranscriptional regulatory events.

Small RNA regulation of reproductive function

Post‐transcriptional gene regulation is one mechanism that occurs “above the genome,” allowing the cells of an organism to have dramatically different phenotypes and functions. Non‐coding ribonucleic acid (ncRNA) molecules regulate transcript and protein abundance above the level of transcription, and appear to play substantial roles in regulation of reproductive tissues. Three primary classes of small ncRNA are microRNA (miRNA), endogenous small interfering RNA (endo‐siRNA), and PIWI‐interacting RNA (piRNA). These RNA classes have similarities and clear distinctions between their biogenesis and in the interacting protein machinery that facilitate their effects on cellular phenotype. Characterization of the expression and importance of the critical components for the biogenesis of each class in different tissues is continuously contributing a better understanding of each of these RNA classes in different reproductive cell types. Here, we discuss the expression and potential roles of miRNA, endo‐siRNA, and piRNA in reproduction from germ‐cell development to pregnancy establishment and placental function. Additionally, the potential contribution of RNA binding proteins, long ncRNAs, and the more recently discovered circular RNAs (circRNAs) in relation to small RNA function is discussed. Mol. Reprod. Dev. 81: 148–159, 2014.

Expression of RNA‐binding proteins DND1 and FXR1 in the porcine ovary, and during oocyte maturation and early embryo development

The porcine oocyte and early embryo are transcriptionally quiescent following germinal vesicle breakdown in the oocyte and prior to activation of the embryonic genome, at approximately the 4‐cell stage of development. Despite a lack of new transcription, mRNA and protein repertoires are subject to regulation during this time. One potential mechanism of regulation is through the functional activity of miRNAs and/or the presence of specific RNA‐binding proteins. Both DND1 (dead end homolog 1) and FXR1 (fragile‐X‐mental retardation‐related protein 1) are RNA‐binding proteins that have been demonstrated to impact miRNA‐mediated, post‐transcriptional gene regulation. The objective was to characterize the presence and the expression changes in DND1 and FXR1 during pig oocyte maturation and early embryo development. DND1 and FXR1 expression were evaluated in oocytes and cumulus cells during meiotic progression and in 4‐cell stage embryos using quantitative RT‐PCR, Western blot analysis, and immunostaining. These data demonstrate DND1 and FXR1 mRNA are expressed in the maturing oocyte and early in vitro‐fertilized embryos, with significantly less DND1 in 4‐cell stage embryos as compared to germinal vesicle and metaphase II‐arrested oocytes. Based on immunohistochemistry, DND1 protein abundance is greater in secondary follicles in comparison to primary and tertiary follicles. Using ribonucleoprotein immunoprecipitation from germinal vesicle‐stage oocytes, DND1 was demonstrated to interact with several mRNAs associated with pluripotency. This work provides a better understanding of the biological relevance of DND1 and FXR1 during female gametogenesis and embryo development in pigs.Mol. Reprod. Dev. 79: 541‐552, 2012.

MicroRNA-21 and PDCD4 expression during in vitro oocyte maturation in pigs

BackgroundMicroRNA (miRNA) are small non-coding RNA molecules critical for regulating cellular function, and are abundant in the maturing oocyte and developing embryo. MiRNA-21 (MIR21) has been shown to elicit posttranscriptional gene regulation in several tissues associated with rapid cell proliferation in addition to demonstrating anti-apoptotic features through interactions with PDCD4 mRNA and other targets. In many tissues, MIR21 interacts and suppresses PDCD4 due to the strong complementation between MIR21 and the PDCD4 3′UTR.MethodsThe objective of this project was to examine the relationship between MIR21 and PDCD4 expression in porcine oocytes during in vitro maturation and assess the impact of MIR21 inhibition during oocyte maturation on early embryo development. Additionally, we evaluated the effect of gonadotropins in maturation media and the presence of cumulus cells to determine their ability to contribute to MIR21 abundance in the oocyte during maturation.ResultsDuring in vitro maturation, expression of MIR21 increased approximately 6-fold in the oocyte and 25-fold in the cumulus cell. Temporally associated with this was the reduction of PDCD4 protein abundance in MII arrested oocytes compared with GV stage oocytes, although PDCD4 mRNA was not significantly different during this transition. Neither the presence of cumulus cells nor gonadotropins during in vitro maturation affected MIR21 abundance in those oocytes achieving MII arrest. However, inhibition of MIR21 activity during in vitro maturation using antisense MIR21 suppressed embryo development to the 4–8 cell stage following parthenogenetic activation.ConclusionsMIR21 is differentially expressed in the oocyte during meiotic maturation in the pig and inhibition of MIR21 during this process alters PDCD4 protein abundance suggesting posttranscriptional regulatory events involving MIR21 during oocyte maturation may impact subsequent embryonic development in the pig.

Novel microRNA families expanded in the human genome

BackgroundMost studies on the origin and evolution of microRNA in the human genome have been focused on its relationship with repetitive elements and segmental duplications. However, duplication events at a smaller scale (<1 kb) could also contribute to microRNA expansion, as demonstrated in this study.ResultsUsing comparative genome analysis and bioinformatics methods, we found nine novel expanded microRNA families enriched in short duplicated sequences in the human genome. Furthermore, novel genomic regions were found to contain microRNA paralogs for microRNA families previously analyzed to be related to segmental duplications. We found that for microRNA families expanded in the human genome, 14 families are specific to the primate lineage, and nine are non-specific, respectively. Two microRNA families (hsa-mir-1233 and hsa-mir-622) appear to be further expanded in the human genome, and were confirmed by fluorescence in situ hybridization. These novel microRNA families expanded in the human genome were mostly embedded in or close to proteins with conserved functions. Furthermore, besides the Alu element, L1 elements could also contribute to the origination of microRNA paralog families.ConclusionsTogether, we found that small duplication events could also contribute to microRNA expansion, which could provide us novel insights on the evolution of human genome structure and function.

Genetic Modification of Domestic Animals for Agriculture and Biomedical Applications

Transgenic technology has been applied mainly in the study of gene structure and function in model organisms and gene therapy for human diseases. Transgenic technology has potential for rapidly improving quantity and quality of agricultural products, compared to traditional selection and breeding methods in domestic animals that are time consuming when attempting to alter the desired allele frequency for specific traits. Additionally, transgenic animals can be used as biomedical research models or directly for human health, by producing recombinant pharmaceutical proteins and/or organs for xenotransplantation. Due to the advantage of bypassing the need of embryonic stem (ES) cells that are difficult to isolate in domestic animal species, cell-based method of transgenesis followed by somatic cell nuclear transfer (SCNT) is currently widely applied. However, due to the limitations in making genetic modifications and SCNT, producing genetically modified animals is still inefficient. Fortunately, the current advancement of new techniques and methods in both gene targeting (Urnov et al., 2010) and abilities to produce pluripotent stem cells (Voigt and Serikawa, 2009) holds great promises for this field. In this chapter, we will review the recent progress and technical route of the cell-based method of transgenesis by SCNT and discuss the newly emerging methods to enrich the gene targeting frequency of somatic cells. We will also discuss factors to improve the efficiency of SCNT and our future perspectives on the promises of this field.