Background: MicroRNAs are the major class of gene-regulating molecules playing diverse roles through sequence complementarity to target mRNAs at post-transcriptional level. Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis could be regulated by these miRNAs. Identification of them is the first step towards understanding miRNA-guided gene regulation in different biological functions. Despite increasing efforts in miRNAs identification across various species and diverse tissue types, little is known about bovine ovarian miRNAs. Here, we report the identification and characterization of miRNAs expressed in the bovine ovary through cloning, expression analysis and target prediction. Results: The miRNA library (5'-independent ligation cloning method), which was constructed from bovine ovary in this study, revealed cloning of 50 known and 24 novel miRNAs. Among all identified miRNAs, 38 were found to be new for bovine and were derived from 43 distinct loci showing characteristic secondary structure. While 22 miRNAs precursor loci were found to be well conserved in more than one species, 16 were found to be bovine specific. Most of the miRNAs were cloned multiple times, in which let-7a, let-7b, let-7c, miR-21, miR-23b, miR-24, miR-27a, miR-126 and miR-143 were cloned 10, 28, 13, 4, 11, 7, 6, 4 and 11 times, respectively. Expression analysis of all new and some annotated miRNAs in different intra-ovarian structures and in other multiple tissues showed that some were present ubiquitously while others were differentially expressed among different tissue types. Bta-miR-29a was localized in the follicular cells at different developmental stages in the cyclic ovary. Bio-informatics prediction, screening and Gene Ontology analysis of miRNAs targets identified several biological processes and pathways underlying the ovarian function. Conclusion: Results of this study suggest the presence of miRNAs in the bovine ovary, thereby elucidate their potential role in regulating diverse molecular and physiological pathways underlying the ovarian functionality. This information will give insights into bovine ovarian miRNAs, which can be further characterized for their role in follicular development and female fertility as well.

Background: The bi-directional communication between the oocyte and its companion cumulus cells (CCs) is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa. Results: We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV) oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII) oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO + CCs) and without (OO - CCs) CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs + OO) or without (CCs - OO) their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs + OO and CCs - OO, respectively. While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18), translation (EIF2AK1, EIF4ENIF1) and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI), protein metabolic processes (IHH, APOA1, PLOD1), steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7). Similarly, while transcripts over expressed in OO + CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs + OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2). Conclusion: In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes. Moreover, the presence or absence of oocyte and CC factors during bovine oocyte maturation can have a profound effect on transcript abundance of each cell types, thereby showing the prevailing molecular cross-talk between oocytes and their corresponding CCs.

The genetic codes inscribed during two key developmental processes, namely gametogenesis and embryogenesis, are believed to determine subsequent development and survival of adult life. Once the embryo is formed, its further development mainly depends on its intrinsic characteristics, maternal environment (the endometrial receptivity), and the embryo–maternal interactions established during each phase of development. These developmental processes are under strict genetic regulation that could be manifested temporally and spatially depending on the physiological and developmental status of the cell. MicroRNAs (miRNAs), one of the small non-coding classes of RNAs, approximately 19–22 nucleotides in length, are one of the candidates for post-transcriptional developmental regulators. These tiny non-coding RNAs are expressed in ovarian tissue, granulosa cells, testis, oocytes, follicular fluid, and embryos and are implicated in diverse biological processes such as cell-to-cell communication. Moreover, accumulated evidences have also highlighted that miRNAs can be released into the extracellular environment through different mechanisms facilitating intercellular communication. Therefore, understanding miRNAs mediated regulatory mechanisms during gametogenesis and embryogenesis provides further insights about the molecular mechanisms underlying oocyte/sperm formation, early embryo development, and implantation. Thus, this review highlights the role of miRNAs in mammalian gametogenesis and embryogenesis and summarizes recent findings about miRNA-mediated post-transcriptional regulatory mechanisms occurring during early mammalian development.

Clinical and subclinical endometritis are known to affect the fertility of dairy cows by inducing uterine inflammation. We hypothesized that clinical or subclinical endometritis could affect the fertility of cows by disturbing the molecular milieu of the uterine environment. Here we aimed to investigate the endometrial molecular signatures and pathways affected by clinical and subclinical endometritis. For this, Holstein Frisian cows at 42–60 days postpartum were classified as healthy (HE), subclinical endometritis (SE) or clinical endometritis (CE) based on veterinary clinical examination of the animals and histological evaluation the corresponding endometrial biopsies. Endometrial transcriptome and miRNome profile changes and associated molecular pathways induced by subclinical or clinical endometritis were then investigated using GeneChip® Bovine Genome Array and Exiqon microRNA PCR Human Panel arrays, respectively. The results were further validated in vitro using endometrial stromal and epithelial cells challenged with subclinical and clinical doses of lipopolysaccharide (LPS). Transcriptome profile analysis revealed altered expression level of 203 genes in CE compared to HE animals. Of these, 92 genes including PTHLH, INHBA, DAPL1 and SERPINA1 were significantly upregulated, whereas the expression level of 111 genes including MAOB, CXCR4, HSD11B and, BOLA, were significantly downregulated in CE compared to the HE animal group. However, in SE group, the expression patterns of only 28 genes were found to be significantly altered, of which 26 genes including PTHLH, INHBA, DAPL1, MAOB, CXCR4 and TGIF1 were common to the CE group. Gene annotation analysis indicated the immune system processes; G-protein coupled receptor signaling pathway and chemotaxis to be among the affected functions in endometritis animal groups. In addition, miRNA expression analysis indicated the dysregulation of 35 miRNAs including miR-608, miR-526b* and miR-1265 in CE animals and 102 miRNAs including let-7 family (let-7a, let-7c, let-7d, let-7d*, let-7e, let-7f, let-7i) in SE animals. Interestingly, 14 miRNAs including let-7e, miR-92b, miR-337-3p, let-7f and miR-145 were affected in both SE and CE animal groups. Further in vitro analysis of selected differentially expressed genes and miRNAs in endometrial stroma and epithelial cells challenged with SE and CE doses of LPS showed similar results to that of the array data generated using samples collected from SE and CE animals. The results of this study unraveled endometrial transcriptome and miRNome profile alterations in cows affected by subclinical or clinical endometritis which may have a significant effect on the uterine homeostasis and uterine receptivity.

Gene expression analysis using real-time RT-PCR (qRT-PCR) is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR. For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes or internal control genes is required. The stability of reference genes has a tremendous effect on the results of relative quantification of gene expression by qRT-PCR. The expression stability of reference genes could vary according to tissues, age of individuals and experimental conditions. In the pig however, very little information is available on the expression stability of reference genes. The aim of this research was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in varieties of porcine tissues at different ages.

Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H₂O₂ by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage.