Characterization of bovine long non-coding RNAs, OOSNCR1, OOSNCR2 and OOSNCR3, and their roles in oocyte maturation and early embryonic development.

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

Characterization of agouti-signaling protein (ASIP) in the bovine ovary and throughout early embryogenesis.

The oocyte expresses certain genes during folliculogenesis to regulate the acquisition of oocyte competence. Oocyte competence, or oocyte quality, is directly related to the ability of the oocyte to result in a successful pregnancy following fertilization. Presently, approximately 40 % of bovine embryos will develop to the blastocyst stage in vitro. Characterization of factors regulating these processes is crucial to improve the efficiency of bovine in vitro embryo production. We demonstrated that the secreted protein, agouti-signaling protein (ASIP) is highly abundant in the bovine oocyte and aimed to characterize its spatiotemporal expression profile in the ovary and throughout early embryonic development. In addition to oocyte expression, ASIP was detected in granulosa, cumulus, and theca cells isolated from antral follicles. Both gene expression data and immunofluorescent staining indicated ASIP declines with oocyte maturation which may indicate a potential role for ASIP in the attainment of oocyte competence. Microinjection of zygotes using small interfering RNA targeting ASIP led to a 16 % reduction in the rate of development to the blastocyst stage. Additionally, we examined potential ASIP signaling mechanisms through which ASIP may function to establish oocyte developmental competence. The expression of melanocortin receptor 3 and 4 and the coreceptor attractin was detected in the oocyte and follicular cells. The addition of cortisol during in vitro maturation was found to increase significantly oocyte ASIP levels. In conclusion, these results suggest a functional role for ASIP in promoting oocyte maturation and subsequent embryonic development, potentially through signaling mechanisms involving cortisol.

Identification of the DNA binding element of ZNFO, an oocyte-specific zinc finger transcription factor in cattle.

The maternal effect genes are essential components of oocyte competence, which orchestrate the early developmental events before zygotic genome activation (ZGA). The Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KRAB-ZFPs) constitute the largest transcription factor family in mammals. As a novel maternal effect gene, ZNFO was identified previously in our laboratory. The gene codes for a KRAB-ZFP specifically expressed in bovine oocytes and early embryos and gene silencing experiments have demonstrated that ZNFO is required for early embryonic development in cattle. In the present study, we identified a consensus sequence, ATATCCTGTTTAAACCCC, as the DNA binding element of ZNFO (ZNFOBE) using a library of random oligonucleotides by cyclic amplification of sequence target (CAST) analysis. Sequence-specific binding of ZNFO to the DNA binding element was confirmed by an electrophoretic mobility shift assay (EMSA), and the key nucleotides in the ZNFOBE that are required for specific binding by ZNFO were further determined by a competitive EMSA using mutant competitors. Through a luciferase-based reporter assay, it was confirmed that the interaction between ZNFO and ZNFOBE is required for the repressive function of ZNFO. These results provide an essential step towards the identification of ZNFO regulated genes that play important roles during early embryonic development.

Galectin-1 induces gene and protein expression related to maternal-conceptus immune tolerance in bovine endometrium†.

Conceptus secretory factors include galectins, a family of carbohydrate binding proteins that elicit cell adhesion and immune suppression by interacting with intracellular and extracellular glycans. In rodents, galectin-1 (LGALS1) promotes maternal-fetal immune tolerance in the decidua through expansion of tolerogenic cluster of differentiation 11c (CD11c) positive dendritic cells, increased anti-inflammatory interleukin (IL)-10, and activation of forkhead box P3 (FOXP3) positive regulatory T cells (Treg). This study characterized galectin expression in early ruminant conceptuses and endometrium. We also tested the effect of recombinant bovine LGALS1 (rbLGALS1) and progesterone (P4) on endometrial expression of genes and protein related to maternal-conceptus immune tolerance in cattle. Elongating bovine and ovine conceptuses expressed several galectins, particularly, LGALS1, LGALS3, and LGALS8. Within bovine endometrium, expression of LGALS3, LGALS7, and LGALS9 was greater on Day 16 of pregnancy compared to the estrous cycle. Within ovine endometrium, LGALS7 was greater during pregnancy compared to the estrous cycle and endometrium of pregnant sheep tended to have greater LGALS9 and LGALS15. Expression of endometrial LGALS4 was less during pregnancy in sheep. Treating bovine endometrium with rbLGALS1 increased endometrial expression of CD11c, IL-10, and FOXP3, within 24 h. Specifically, within caruncular endometrium, both rbLGALS1 and P4 increased FOXP3, suggesting that both ligands may promote Treg expansion. Using IHC, FOXP3+ cells with a leukocyte phenotype were localized to the bovine uterine stratum compactum near the uterine surface and increased in response to rbLGALS1. We hypothesize that galectins have important functions during establishment of pregnancy in ruminants and bovine conceptus LGALS1 and luteal P4 confer mechanisms of maternal-conceptus immune tolerance in cattle.

Identification of the core promoter of ZNFO, an oocyte-specific maternal effect gene in cattle.

ZNFO is a Krüppel-associated box (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocytes. Previous studies have demonstrated that ZNFO possesses an intrinsic transcriptional repressive activity and is essential for early embryonic development in cattle. However, the mechanisms regulating ZNFO transcription remain elusive. In the present study, the core promoter that controls the ZNFO basal transcription was identified. A 1.7 kb 5' regulatory region of the ZNFO gene was cloned and its promoter activity was confirmed by a luciferase reporter assay. A series of 5' deletion in the ZNFO promoter followed by luciferase reporter assays indicated that the core promoter region has to include the sequence located within 57 bp to 31 bp upstream of the transcription start site. Sequence analysis revealed that a putative USF1/USF2 binding site (GGTCACGTGACC) containing an E-box motif (CACGTG) is located within the essential region. Depletion of USF1/USF2 by RNAi and E-box mutation analysis demonstrated that the USF1/USF2 binding site is required for the ZNFO basal transcription. Furthermore, EMSA and super-shift assays indicated that the observed effects are dependent on the specific interactions between USF proteins and the ZNFO core promoter. From these results, it is concluded that USF1 and USF2 are essential for the basal transcription of the ZNFO gene.

Conceptus-induced, interferon tau-dependent gene expression in bovine endometrial epithelial and stromal cells†.

Bovine endometrium consists of epithelial and stromal cells that respond to conceptus interferon tau (IFNT), the maternal recognition of pregnancy (MRP) signal, by increasing expression of IFN-stimulated genes (ISGs). Endometrial epithelial and stromal-cell-specific ISGs are largely unknown but hypothesized to have essential functions during pregnancy establishment. Bovine endometrial epithelial cells were cultured in inserts above stromal fibroblast (SF) cells for 6 h in medium alone or with IFNT. The epithelial and SF transcriptomic response was analyzed separately using RNA sequencing and compared to a list of 369 DEGs recently identified in intact bovine endometrium in response to elongating bovine conceptuses and IFNT. Bovine endometrial epithelial and SF shared 223 and 70 DEGs in common with the list of 369 endometrial DEGs. Well-known ISGs identified in the epithelial and SF were ISG15, MX1, MX2, and OAS2. DEGs identified in the epithelial but not SF included a number of IRF molecules (IRF1, IRF2, IRF3, and IRF8), mitochondria SLC transporters (SLC25A19, SLC25A28, and SLC25A30), and a ghrelin receptor. Expression of ZC3HAV1, an anti-retroviral gene, increased specifically within the SF. Gene ontology analysis identified the type I IFN signaling pathway and activation of nuclear factor kappa B transcription factors as biological processes associated with the epithelial cell DEGs. This study has identified biologically relevant IFNT-stimulated genes within specific endometrial cell types. The findings provide critical information regarding the effects of conceptus IFNT on specific endometrial compartments during early developmental processes in cattle.