Granulosa cells undergo BPA-induced apoptosis in a miR-21-independent manner.

Bisphenol A (BPA) is a harmful endocrine disrupting compound that alters not only classical cellular mechanisms but also epigenetic mechanisms. Evidence suggests that BPA-induced changes in microRNA expression can explain, in part, the changes observed at both the molecular and cellular levels. BPA is toxic to granulosa cells (GCs) as it can activate apoptosis, which is known to contribute to increased follicular atresia. miR-21 is a crucial antiapoptotic regulator in GCs, yet the exact function in a BPA toxicity model remains unclear. BPA was found to induce bovine GC apoptosis through the activation of several intrinsic factors. BPA reduced live cells counts, increased late apoptosis/necrosis, increased apoptotic transcripts (BAX, BAD, BCL-2, CASP-9, HSP70), increased the BAX/Bcl-2 ratio and HSP70 at the protein level, and induced caspase-9 activity at 12 h post-exposure. miR-21 inhibition increased early apoptosis and, while it did not influence transcript levels or caspase-9 activity, it did elevate the BAX/Bcl-2 protein ratio and HSP70 in the same manner as BPA. Overall, this study shows that miR-21 plays a molecular role in regulating intrinsic mitochondrial apoptosis; however, miR-21 inhibition did not make the cells more sensitive to BPA. Therefore, apoptosis induced by BPA in bovine GCs is miR-21 independent.

cDNA Cloning of Feline PIWIL1 and Evaluation of Expression in the Testis of the Domestic Cat.

The PIWI clade of Argonaute proteins is essential for spermatogenesis in all species examined to date. This protein family binds specific classes of small non-coding RNAs known as PIWI-interacting RNAs (piRNAs) which together form piRNA-induced silencing complexes (piRISCs) that are recruited to specific RNA targets through sequence complementarity. These complexes facilitate gene silencing through endonuclease activity and guided recruitment of epigenetic silencing factors. PIWI proteins and piRNAs have been found to play multiple roles in the testis including the maintenance of genomic integrity through transposon silencing and facilitating the turnover of coding RNAs during spermatogenesis. In the present study, we report the first characterization of PIWIL1 in the male domestic cat, a mammalian system predicted to express four PIWI family members. Multiple transcript variants of PIWIL1 were cloned from feline testes cDNA. One isoform shows high homology to PIWIL1 from other mammals, however, the other has characteristics of a "slicer null" isoform, lacking the domain required for endonuclease activity. Expression of PIWIL1 in the male cat appears limited to the testis and correlates with sexual maturity. RNA-immunoprecipitation revealed that feline PIWIL1 binds small RNAs with an average size of 29 nt. Together, these data suggest that the domestic cat has two PIWIL1 isoforms expressed in the mature testis, at least one of which interacts with piRNAs.

BPA Decreases PDCD4 in Bovine Granulosa Cells Independently of miR-21 Inhibition.

microRNAs (miRNAs) are susceptible to environmental factors that might affect cellular function and impose negative effects on female reproduction. miR-21 is the most abundant miRNA in bovine granulosa cells and is widely reported as affected by Bisphenol A (BPA) exposure, yet the cause and consequences are not entirely elucidated. BPA is a synthetic endocrine disruptor associated with poor fertility. miR-21 function in bovine granulosa cells is investigated utilizing locked nucleic acid (LNA) oligonucleotides to suppress miR-21. Before measuring apoptosis and quantifying miR-21 apoptotic targets PDCD4 and PTEN, transfection was optimized and validated. BPA was introduced to see how it affects miR-21 regulation and which BPA-mediated effects are influenced by miR-21. miR-21 knockdown and specificity against additional miRNAs were confirmed. miR-21 was found to have antiapoptotic effects, which could be explained by its effect on the proapoptotic target PDCD4, but not PTEN. Previous findings of miR-21 overexpression were validated using BPA treatments, and the temporal influence of BPA on miR-21 levels was addressed. Finally, BPA effects on upstream regulators, such as VMP1 and STAT3, explain the BPA-dependent upregulation of miR-21 expression. Overall, this research enhances our understanding of miR-21 function in granulosa cells and the mechanisms of BPA-induced reproductive impairment.

In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos.

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.

Why the hype - What are microRNAs and why do they provide unique investigative, diagnostic, and therapeutic opportunities in veterinary medicine?

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by inhibiting translation or inducing transcript degradation. MiRNAs act as fine-tuning factors that affect the expression of up to 60% of all mammalian protein coding genes. In contrast to proteins, there is widespread conservation of miRNA sequences across species. This conservation strongly suggests that miRNAs appeared early in evolution and have retained their functional importance. Cross-species conservation provides advantages when compiling candidate markers for health and disease compared to protein-based discoveries. This broad utility is accompanied by the emergence of inexpensive sequencing protocols for the identification of all RNAs in a sample (including miRNAs). With the use of miRNA mimics and antagonists, unique research questions can be answered in biological systems with 'cause and effect' methodology. MiRNAs are readily detectable in blood making them attractive candidates as biomarkers for disease. Here, we review their utility as biomarkers and their potential as therapeutic agents or targets to combat disease.

Pourquoi la frénésie ­ Que sont les microRNAs et pourquoi fournissent-ils des opportunités uniques pour investiguer, diagnostiquer et traiter en médecine vétérinaire? Les microRNAs (MiRNAs) sont de petits segments non-codants d'ARN qui régulent l'expression des gènes en inhibant la traduction ou en induisant la dégradation du transcript. Les MiRNAs agissent comme des facteurs d'ajustement fin qui affectent l'expression pouvant aller jusqu'à 60 % de tous les gènes mammaliens codant pour des protéines. Contrairement aux protéines, il y a un conservatisme étendu des séquences des miRNA à travers les espèces. Ce conservatisme suggère fortement que les miRNAs sont apparus tôt dans l'évolution et ont conservé leur importance fonctionnelle. La conservation inter-espèces fournie des avantages lors de la compilation de candidats marqueurs pour la santé et la maladie comparativement aux découvertes basées sur les protéines. Cette large utilité est accompagnée par l'émergence de protocoles de séquençage peu dispendieux pour l'identification de tous les ARNs dans un échantillon (incluant miRNAs). Avec l'utilisation d'imitations et d'antagonistes de miRNA, des questionnements rares en recherche peuvent être répondus dans des systèmes biologiques avec des méthodologies « cause et effet ¼. Les miRNAs sont facilement détectables dans le sang ce qui les rend des candidats attirants comme biomarqueurs de maladies. Ici, nous faisons une revue de leur utilité comme biomarqueurs et leur potentiel comme agents thérapeutiques ou cibles pour combattre des maladies.(Traduit par Dr Serge Messier).

MicroRNAs: potential targets and agents of endocrine disruption in female reproduction.

MicroRNAs are short non-coding RNAs that have been widely recognized as key mediators in the epigenetic control of gene expression and which are present in virtually all cells and tissues studied. These regulatory molecules are generated in multiple steps in a process called microRNA biogenesis. Distinct microRNA expression patterns during the different stages of oocyte and embryo development suggest important regulatory roles for these small RNAs. Moreover, studies antagonizing specific microRNAs and enzymes in microRNA biogenesis pathways have demonstrated that interference with normal miRNA function leads to infertility and is associated with some reproductive abnormalities. Endocrine disrupting chemicals such as Bisphenol A (BPA) are synthetic hormone mimics that have been found to negatively impact reproductive health. In addition to their direct effects on gene expression, these chemicals are widely implicated in the disruption of epigenetic pathways, including the expression and activity of miRNAs, thereby altering gene expression. In this review, the roles of microRNAs during mammalian oocyte and embryo development are outlined and the different mechanisms by which endocrine disruptors such as BPA interfere with these epigenetic regulators to cause reproductive problems is explored.

Transposons and the PIWI pathway: genome defense in gametes and embryos

Hiding in plain sight within the genome of virtually every eukaryotic organism are large numbers of sequences known as transposable elements (TEs). These sequences often comprise 50% or more of the DNA in many mammals and are transcriptionally constrained by DNA methylation and repressive chromatin marks. Individual TEs, when relieved of these epigenetic constraints, can readily move from one genomic location to another, either directly or through RNA intermediates. Demethylation and removal of repressive histone marks during epigenetic reprogramming stages of gametogenesis and embryogenesis render the genome particularly susceptible to increased TE mobilization, which has significant implications for the fidelity of genome replication and subsequent viability of the progeny. Importantly, however, TEs have functionally integrated themselves into developmental events to the extent that complete suppression precludes normal gamete and embryo development. Consequently, multiple mechanisms have evolved to limit the extent of TE expression and mobilization during reprogramming without completely suppressing it. One of the most important TE repression mechanisms is the PIWI/piRNA pathway, in which 25­32 nucleotide RNA molecules known as piRNAs associate with Argonaute proteins from the PIWI clade to form piRISC complexes. These complexes target and silence TEs post-transcriptionally and through the induction of epigenetic changes at the loci from which they are expressed. This review will briefly discuss the intricate molecular détente between TE expression and its suppression by the PIWI pathway, with particular emphasis on mammalian species including human, bovine and murine.

Disrupting the key circadian regulator CLOCK leads to age-dependent cardiovascular disease.

The circadian mechanism underlies daily rhythms in cardiovascular physiology and rhythm disruption is a major risk factor for heart disease and worse outcomes. However, the role of circadian rhythms is generally clinically unappreciated. Clock is a core component of the circadian mechanism and here we examine the role of Clock as a vital determinant of cardiac physiology and pathophysiology in aging. ClockΔ19/Δ19 mice develop age-dependent increases in heart weight, hypertrophy, dilation, impaired contractility, and reduced myogenic responsiveness. Young ClockΔ19/Δ19 hearts express dysregulated mRNAs and miRNAs in the PTEN-AKT signal pathways important for cardiac hypertrophy. We found a rhythm in the Pten gene and PTEN protein in WT hearts; rhythmic oscillations are lost in ClockΔ19/Δ19 hearts. Changes in PTEN are associated with reduced AKT activation and changes in downstream mediators GSK-3ß, PRAS40, and S6K1. Cardiomyocyte cultures confirm that Clock regulates the AKT signalling pathways crucial for cardiac hypertrophy. In old ClockΔ19/Δ19 mice cardiac AKT, GSK3ß, S6K1 phosphorylation are increased, consistent with the development of age-dependent cardiac hypertrophy. Lastly, we show that pharmacological modulation of the circadian mechanism with the REV-ERB agonist SR9009 reduces AKT activation and heart weight in old WT mice. Furthermore, SR9009 attenuates cardiac hypertrophy in mice subjected to transverse aortic constriction (TAC), supporting that the circadian mechanism plays an important role in regulating cardiac growth. These findings demonstrate a crucial role for Clock in growth and renewal; disrupting Clock leads to age-dependent cardiomyopathy. Pharmacological targeting of the circadian mechanism provides a new opportunity for treating heart disease.

CpG oligonucleotide-mediated co-stimulation of mouse invariant natural killer T cells negatively regulates their activation status.

Invariant natural killer T (iNKT) cells play important roles in antimicrobial defense and immune-regulation. We have previously shown that iNKT cells express certain toll-like receptors (TLR), and that TLR co-stimulation of iNKT cells in the presence of suboptimal concentrations of T cell receptor (TCR) agonists enhances cellular activation. In the present study, we investigated the regulatory effects of CpG oligonucleotides in mouse primary hepatic and splenic iNKT cells and in DN32.D3 iNKT cells. We show that CpG treatment of iNKT cells in the presence of higher concentrations of TCR agonists (α-GalCer or anti-CD3 mAb) results in the up-regulation of TLR9 in iNKT cells with a concurrent reduction in their cellular activation, as assessed by their production of IL-2, IL-4 and IFN-γ compared with controls. CpG-mediated down-regulation of iNKT cell activation has been found to depend, at least in part, on signaling by MyD88, a critical adapter moiety downstream of TLR9 signaling. Mechanistically, iNKT cells treated with CpG in the presence of TCR agonists show inhibition of MAPK signaling as determined by the levels of ERK1/2 and p38 MAPKs. Furthermore, CpG treatment leads to an increased induction of phosphatases, DUSP1 and SHP-1, that seem to impede MAPK and TCR signaling, resulting in the negative regulation of iNKT cell activation. Our findings therefore suggest a novel regulatory role for CpG in iNKT cells in the mediation of a negative feedback mechanism to control overactive iNKT cell responses and hence to avoid undesirable excessive immunopathology.

Bovine piRNA-like RNAs are associated with both transposable elements and mRNAs.

PIWI proteins and their associated piRNAs have been the focus of intensive research in the past decade; therefore, their participation in the maintenance of genomic integrity during spermatogenesis has been well established. Recent studies have suggested important roles for the PIWI/piRNA system outside of gametogenesis, based on the presence of piRNAs and PIWI proteins in several somatic tissues, cancers, and the early embryo. Here, we investigated the small RNA complement present in bovine gonads, gametes, and embryos through next-generation sequencing. A distinct piRNA population was present in the testis as expected. However, we also found a large population of slightly shorter, 24-27 nt piRNA-like RNA (pilRNAs) in pools of oocytes and zygotes. These oocyte and embryo pilRNAs exhibited many of the canonical characteristics of piRNAs including a 1U bias, the presence of a 'ping-pong' signature, genomic clustering, and transposable element targeting. Some of the major transposons targeted by oocyte and zygote pilRNA were from the LINE RTE and ERV1 classes. We also identified pools of pilRNA potentially derived from, or targeted at, specific mRNA sequences. We compared the frequency of these gene-associated pilRNAs to the fold change in the expression of respective mRNAs from two previously reported transcriptome datasets. We observed significant negative correlations between the number of pilRNAs targeting mRNAs, and their fold change in expression between the 4-8 cell and 8-16 cell stages. Together, these results represent one of the first characterizations of the PIWI/piRNA pathway in the translational bovine model, and in the novel context of embryogenesis.

PIWIL1 Is Expressed in the Canine Testis, Increases with Sexual Maturity, and Binds Small RNAs.

Spermatogenesis is a highly regulated process leading to the development of functional spermatozoa through meiotic division and subsequent maturation. Recent studies have suggested that a novel class of Argonaute proteins, known as the PIWI clade, plays important roles in multiple stages of spermatogenesis. PIWI proteins bind specific small noncoding RNAs, called PIWI-interacting RNAs (piRNAs). These piRNAs guide the PIWI-piRNA complex to retrotransposon targets that become expressed during meiosis. Retrotransposons are subsequently silenced, either through PIWI "slicer" activity or through PIWI-directed methylation of the retrotransposon locus. Most mammalian studies have employed mouse models where sterility follows PIWI inactivation. The goal of this study was to characterize canine PIWIL1 to determine whether expression pattern and functional characteristics support a similar function in that species. Canine PIWIL1 cDNA is a 2.6-kb transcript that encodes an 861-amino acid protein showing high homology to other mammalian PIWIL1 proteins and containing features consistent with PIWI family members (PAZ, PIWI domains). Analysis of PIWIL1 protein and transcript levels revealed that PIWIL1 expression is limited to the testes and is associated with sexual maturity, with mature dogs showing higher levels of PIWIL1 expression. Immunohistochemistry demonstrated expression primarily in seminiferous tubules and confirmed higher levels of PIWIL1 in mature dogs. Functional characterization by RNA immunoprecipitation demonstrated that canine PIWIL1 binds short RNAs consistent in size with piRNAs (27-32 nucleotides). Together, these studies represent the first characterization of a PIWI protein in the dog and suggest that it is a functional piRNA-binding protein most highly expressed in the mature testes.

Identification of PIWIL1 Isoforms and Their Expression in Bovine Testes, Oocytes, and Early Embryos.

PIWI proteins are members of the larger Argonaute family and bind to specific 24-32 nucleotide RNAs called PIWI-interacting RNAs (piRNAs). PIWI-interacting RNAs direct PIWI-mediated suppression of retrotransposon expression in the male germline in humans and mice, but their roles in bovine reproduction and embryogenesis are unknown. Although the majority of research in mammals has focused on the functions of PIWI proteins during spermatogenesis, this family of proteins and their associated piRNAs have recently been identified in early embryos. The goals of this study were to characterize the expression of PIWIL1 in bovine testis, oocytes, and early embryos. A full-lengthPIWIL1transcript and protein was found in the testis, specifically in the germs cells of mature seminiferous tubules. RNA-immunoprecipitation demonstrated the presence of putative piRNAs with a mean length of 30 nucleotides bound to PIWIL1 in testes. 3'-Rapid amplification of cDNA ends analysis ofPIWIL1transcripts in testes and oocytes revealed two shorter isoforms in addition to the full-length transcript that was only present in testes. TruncatedPIWIL1isoforms in oocytes and testes were confirmed through amplification of their unique intronic fragments. Expression profiling ofPIWIL1through early embryogenesis demonstrated peak mRNA expression at the 2-cell stage with decreasing levels through to the blastocyst. PIWIL1-YFP fusion plasmids were produced for each isoform and expressed in HEK 293 cells, demonstrating nuclear exclusion and size-specific banding of the different isoforms. These data represent the first comprehensive characterization of PIWIL1 in bovine, revealing functional similarities with PIWIL1 in other species and suggest tissue-specific expression of several isoforms.

An in vitro method to test the safety and efficacy of low-level laser therapy (LLLT) in the healing of a canine skin model.

BACKGROUND: Low-level laser therapy (LLLT) has been used clinically as a treatment modality for a variety of medical conditions including wound-healing processes. It is an attractive and emerging method to enhance wound healing and improve clinical outcomes both in human and veterinary medicine. Despite the fact that the use of LLLT continues to gain in popularity, there is no universally accepted theory that defends all its cellular effects and beneficial biological processes in tissue repair. The present study was designed to evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK) in an in vitro wound healing model. RESULTS: Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. Fifteen independent replicates were performed for each assay. Canine epidermal keratinocyte cells exposed to LLLT with 0.1, 0.2, and 1.2 J/cm(2) migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < 0.0001) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm(2). Irradiation with 10 J/cm(2) was characterized by decreased cellular migration and proliferation. These results revealed that LLLT has a measurable, dose-dependent effect on two different aspects of keratinocyte biology in vitro. CONCLUSION: In this in vitro wound-healing model, LLLT increased cellular migration and proliferation at doses of 0.1, 0.2, and 1.2 J/cm(2) while exposure to 10 J/cm(2) decreased cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior.

An interplay between the p38 MAPK pathway and AUBPs regulates c-fos mRNA stability during mitogenic stimulation.

Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.

MicroRNA Expression during Bovine Oocyte Maturation and Fertilization.

Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.

The ART of selecting the best embryo: A review of early embryonic mortality and bovine embryo viability assessment methods.

Animal reproductive biotechnology is continually evolving. Significant advances have been made in our understanding of early embryonic mortality and embryo development in domestic animals, which has improved the selection and success of in vitro technologies. Yet our knowledge is still relatively limited such that identifying a single embryo with the highest chance of survival and development for transfer remains challenging. While invasive methods such as embryo biopsy can provide useful information regarding the genetic status of the embryos, morphological assessment remains the most common evaluation. A recent shift, however, favors alternative, adjunct approaches for non-invasive assessment of an embryo's viability and developmental potential. Various analytical techniques have facilitated the evaluation of cellular health through the metabolome, the assessment of end products of cellular metabolism, or by analyzing spent media for small RNAs. This review discusses the application of noninvasive approaches for ascertaining the health and viability of in vitro-produced bovine embryos. A comparative analysis of noninvasive techniques for embryo assessment currently being investigated in cattle and humans is also discussed.

MicroRNA-34 family expression in bovine gametes and preimplantation embryos.

BACKGROUND: Oocyte fertilization and successful embryo implantation are key events marking the onset of pregnancy. In sexually reproducing organisms, embryogenesis begins with the fusion of two haploid gametes, each of which has undergone progressive stages of maturation. In the final stages of oocyte maturation, minimal transcriptional activity is present and regulation of gene expression occurs primarily at the post-transcriptional level. MicroRNAs (miRNA) are potent effectors of post-transcriptional gene silencing and recent evidence demonstrates that the miR-34 family of miRNA are involved in both spermatogenesis and early events of embryogenesis. METHODS: The profile of miR-34 miRNAs has not been characterized in gametes or embryos of Bos taurus. We therefore used quantitative reverse transcription PCR (qRT-PCR) to examine this family of miRNAs: miR-34a, -34b and -34c as well as their precursors in bovine gametes and in vitro produced embryos. Oocytes were aspirated from antral follicles of bovine ovaries, and sperm cells were isolated from semen samples of 10 bulls with unknown fertility status. Immature and in vitro matured oocytes, as well as cleaved embryos, were collected in pools. Gametes, embryos and ovarian and testis tissues were purified for RNA. RESULTS: All members of the miR-34 family are present in bovine spermatozoa, while only miR-34a and -34c are present in oocytes and cleaved (2-cell) embryos. Mir-34c demonstrates variation among different bulls and is consistently expressed throughout oocyte maturation and in the embryo. The primary transcript of the miR-34b/c bicistron is abundant in the testes and present in ovarian tissue but undetectable in oocytes and in mature spermatozoa. CONCLUSIONS: The combination of these findings suggest that miR-34 miRNAs may be required in developing bovine gametes of both sexes, as well as in embryos, and that primary miR-34b/c processing takes place before the completion of gametogenesis. Individual variation in sperm miR-34 family abundance may offer potential as a biomarker of male bovine fertility.

The use of alternative polyadenylation sites renders integrin ß1 (Itgb1) mRNA isoforms with differential stability during mammary gland development.

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the ß1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin ß1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.

Interleukin 11 expression in the normal canine eye.

OBJECTIVES: The purpose of this study was to characterize the expression of interleukin-11 (IL-11), a cytokine with anti-inflammatory, cytoprotective, and immune-modulating characteristics, in the canine eye. PROCEDURES: Normal canine eyes were collected from clinically healthy dogs that had been euthanized for reasons unrelated to this study. The distribution of IL-11 expression in the different ocular layers was evaluated by immunofluorescence (eight eyes). Expression levels were quantified (based on fluorescence intensity) using pixel density analysis. Primary cell cultures were derived from all three corneal cell layers. IL-11 mRNA expression was assessed in these cultures using quantitative RT-PCR before and after treatment with TGF-ß1, a known inducer of IL-11 expression. IL-11 protein expression was also assessed in the media of these cells by Western blot analysis. RESULTS: IL-11 protein was detected in the corneal epithelium, keratocytes, and the corneal endothelium of the normal canine eyes examined using immunofluorescence. Baseline IL-11 mRNA expression was noted in the corneal epithelium, fibroblasts, and endothelium using quantitative RT-PCR. Treatment of canine corneal cell lines with TGF-ß1 resulted in statistically significant increases in IL-11 expression in the corneal epithelium, endothelial and fibroblast cell lines with strongest induction noted in the fibroblasts and endothelium. CONCLUSION: This is the first description of IL-11 expression in the canine eye. The protein and mRNA appear to be constitutively present throughout all layers of the cornea and are increased by TGF-ß1, a cytokine important in ocular inflammation and disease.

microRNAs are differentially expressed in equine plasma of horses with osteoarthritis and osteochondritis dissecans versus control horses.

Osteoarthritis (OA) is a leading cause of lameness in horses with no effective disease-modifying treatment and challenging early diagnosis. OA is considered a disease of the joint involving the articular cartilage, subchondral bone, synovial membrane, and ligaments. Osteochondritis dissecans (OCD) is a joint disease consisting of focal defects in the osteochondral unit which may progress to OA later in life. MicroRNAs (miRNAs) have been recognized as small non-coding RNAs that regulate a variety of biological processes and have been detected in biological fluids. MiRNAs are currently investigated for their utility as biomarkers and druggable targets for a variety of diseases. The current study hypothesizes that miRNA profiles can be used to actively monitor joint health and differences in miRNA profiles will be found in healthy vs diseased joints and that differences will be detectable in blood plasma of tested horses. Five horses with OA, OCD, and 4 controls (C) had blood plasma and synovial fluid collected. Total RNA, including miRNA was isolated before generating miRNA libraries from the plasma of the horses. Libraries were sequenced at the Schroeder Arthritis Institute (Toronto). Differential expression analysis was done using DESeq2 and validated using ddPCR. KEGG pathway analysis was done using mirPath v.3 (Diana Tools). 57 differentially expressed miRNAs were identified in OA vs C plasma, 45 differentially expressed miRNAs in OC vs C plasma, and 21 differentially expressed miRNAs in OA vs OCD plasma. Notably, miR-140-5p expression was observed to be elevated in OA synovial fluid suggesting that miR-140-5p may serve as a protective marker early on to attenuate OA progression. KEGG pathway analysis of differentially expressed plasma miRNAs showed relationships with glycan degradation, glycosaminoglycan degradation, and hippo signaling pathway. Interestingly, ddPCR was unable to validate the NGS data suggesting that isomiRs may play an integral role in miRNA expression when assessed using NGS technologies.