MicroRNA Nano-Shuttles: Engineering Extracellular Vesicles as a Cutting-Edge Biotechnology Platform for Clinical Use in Therapeutics.

Extracellular vesicles (EVs) are nano-sized, membranous transporters of various active biomolecules with inflicting phenotypic capabilities, that are naturally secreted by almost all cells with a promising vantage point as a potential leading drug delivery platform. The intrinsic characteristics of their low toxicity, superior structural stability, and cargo loading capacity continue to fuel a multitude of research avenues dedicated to loading EVs with therapeutic and diagnostic cargos (pharmaceutical compounds, nucleic acids, proteins, and nanomaterials) in attempts to generate superior natural nanoscale delivery systems for clinical application in therapeutics. In addition to their well-known role in intercellular communication, EVs harbor microRNAs (miRNAs), which can alter the translational potential of receiving cells and thus act as important mediators in numerous biological and pathological processes. To leverage this potential, EVs can be structurally engineered to shuttle therapeutic miRNAs to diseased recipient cells as a potential targeted 'treatment' or 'therapy'. Herein, this review focuses on the therapeutic potential of EV-coupled miRNAs; summarizing the biogenesis, contents, and function of EVs, as well as providing both a comprehensive discussion of current EV loading techniques and an update on miRNA-engineered EVs as a next-generation platform piloting benchtop studies to propel potential clinical translation on the forefront of nanomedicine.

Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation.

Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from equine FF aspirated in vivo during different stages of follicular development, namely, predeviation (PreDev; 18-20 mm), deviation (Dev; 22-25 mm), postdeviation (PostDev; 26-29 mm), preovulatory (PreOV; 30-35 mm), and impending ovulation (IMP; ∼40 mm). Approximately 176 known miRNAs were found in all groups with 144 mutually detected among all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in expression from the PreDev until the PreOV stage. Another cluster of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression pattern at the PreDev stage until the PostDev stage, with a significant increase at the PreOV stage followed by a significant decrease at the IMP stage. In conclusion, this study provides greater insights into the stage-specific expression dynamics of FF EV-miRNAs during equine follicular development, which may propose novel approaches to improve ART and provide new biomarkers to facilitate the assessment of ovarian pathophysiological conditions.

NRF2-mediated signaling is a master regulator of transcription factors in bovine granulosa cells under oxidative stress condition.

Transcription factors (TFs) are known to be involved in regulating the expression of several classes of genes during folliculogenesis. However, the regulatory role of TFs during oxidative stress (OS) is not fully understood. The current study was aimed to investigate the regulation of the TFs in bovine granulosa cells (bGCs) during exposure to OS induced by H2O2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H2O2 for 40 min. Twenty-four hours later, cells were subjected to various phenotypic and gene expression analyses for genes related to TFs, endoplasmic reticulum stress, apoptosis, cell proliferation, and differentiation markers. The bGCs exhibited higher reactive oxygen species accumulation, DNA fragmentation, and endoplasmic reticulum stress accompanied by reduction of mitochondrial activity after exposure to OS. In addition, higher lipid accumulation and lower cell proliferation were noticed in H2O2-challenged cells. The mRNA level of TFs including NRF2, E2F1, KLF6, KLF9, FOS, SREBF1, SREBF2, and NOTCH1 was increased in H2O2-treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1, were downregulated in the H2O2-challenged group. Moreover, targeted inhibition of NRF2 using small interference RNA resulted in reduced expression of KLF9, FOS, SREBF2, and NOTCH1 genes, while the expression of KLF4 was upregulated. Taken together, bovine granulosa cells exposed to OS exhibited differential expression of various transcription factors, which are mediated by the NRF2 signaling pathway.

Developmental toxicity of lead in rats after gestational exposure and the protective role of taurine.

The present study was conducted to investigate the potential adverse effect of Pb on pregnant Sprague-Dawley rats and their fetuses after maternal exposure, on gestational days (GD) 7-16. The possible protective role of taurine (TA), administered throughout the gestation period (GD 1-20) against Pb toxicity, was also evaluated. Pregnant rats were divided into four groups: Group 1 (control) was given distilled water; Group 2 was exposed to Pb (250 ppm) in drinking water (GD 7-16), whereas Group 3 received TA (50 mg/kg/day) by oral gavage (GD 1-20); Group 4 was exposed to Pb (GD 7-16), whereas pretreated with TA from GD 1 till the end of the gestation period. After termination on GD 20, maternal and embryo-fetal outcomes were evaluated. Blood samples were collected for hematological and biochemical parameters assessment. The results showed that, Pb induced a significant reduction in the maternal body weight, weight gain, uterine and placental weight, in addition to a high incidence of abortion and fetal resorption. Meanwhile, fetuses demonstrated decreased body weight and length, with a high rate of mortality as well as external and skeletal abnormalities. Additionally, Pb induced severe hematological and biochemical alterations in both dams and fetuses. The toxicity of Pb was further emphasized by placental histopathological examination and hepatic DNA fragmentation. Pretreatment with TA greatly attenuated the impact of Pb on both maternal and fetal parameters. Moreover, TA alleviated the incidence of placental damage and hepatic DNA fragmentation. The results highlight the potential prophylaxis role of TA against maternal and developmental Pb toxicity.

Current knowledge and the future potential of extracellular vesicles in mammalian reproduction.

Extracellular vesicles (EVs), which contain various functional classes of vesicles, namely exosomes, microvesicles, and apoptotic bodies, represent the major nano-shuttle to transfer bioactive molecules from donor to recipient cells to facilitate cell-to-cell communication in the follicular, oviduct, and uterine microenvironments. In addition to transferring various molecular cargos in the form of miRNAs, mRNAs, proteins, lipids, and DNA molecules, the relative proportion of those molecular cargos in the reproductive fluids can be associated with the physiological and pathological condition of the host animal. Inside the follicle, EV-mediated circulation of miRNAs has been reported to be associated with the growth status of the enclosed oocytes, the metabolic status, and the advanced maternal aging of the animal. Importantly, EVs have the potential to protect their cargo molecules from extracellular degradation or modification while travelling to the recipient cells. This fact together with the enormous availability in almost all biological fluids and spent culture media make them attractive in the search for biomarkers of oocyte/embryo developmental competence, receptive maternal environment and a multitude of reproductive pathophysiological conditions. One of the key factors that have contributed to the lower efficiency of assisted reproductive technologies (ART) is the absence of several maternal in vivo factors in the ART procedures. For this, several studies have been conducted to supplement various components present in the follicular and oviductal fluids into the existing ART procedures and significant positive impacts have been observed in terms of embryo cleavage rate, blastocyst rate, resistance to stress, and survival after cryopreservation. The potential of EVs in shuttling protective messages against environmental and physiological stressors has been evidenced. The effective use of the EV-coupled molecular signals against stress-associated conditions has the potential to pave the path for the application of these protective signals against oxidative stress-associated pathological conditions including PCOS, ageing, and endometritis. In this review, we provide current knowledge and potential future use of EVs as remedies in reproductive pathophysiological conditions, mainly in follicular and oviductal microenvironments.

The regulatory role of miR-20a in bovine cumulus cells and its contribution to oocyte maturation.

Dynamic changes in microRNAs in oocyte and cumulus cells before and after maturation may explain the spatiotemporal post-transcriptional gene regulation within bovine follicular cells during the oocyte maturation process. miR-20a has been previously shown to regulate proliferation and differentiation as well as progesterone levels in cultured bovine granulosa cells. In the present study, we aimed to demonstrate the function of miR-20a during the bovine oocyte maturation process. Maturation of cumulus-oocyte complexes (COCs) was performed at 39°C in an humidified atmosphere with 5% CO2 in air. The expression of miR-20a was investigated in the cumulus cells and oocytes at 22 h post culture. The functional role of miR-20a was examined by modulating the expression of miR-20a in COCs during in vitro maturation (IVM). We found that the miR-20a expression was increased in cumulus cells but decreased in oocytes after IVM. Overexpression of miR-20a increased the oocyte maturation rate. Even though not statistically significant, miR-20a overexpression during IVM increased progesterone levels in the spent medium. This was further supported by the expression of STAR and CYP11A1 genes in cumulus cells. The phenotypes observed due to overexpression of miR-20a were validated by BMP15 supplementation during IVM and subsequent transfection of BMP15-treated COCs using miR-20a mimic or BMPR2 siRNA. We found that miR-20a mimic or BMPR2 siRNA transfection rescued BMP15-reduced oocyte maturation and progesterone levels. We concluded that miR-20a regulates oocyte maturation by increasing cumulus cell progesterone synthesis by simultaneous suppression of BMPR2 expression.

MicroRNA-Mediated Gene Regulatory Mechanisms in Mammalian Female Reproductive Health.

Mammalian reproductive health affects the entire reproductive cycle starting with the ovarian function through implantation and fetal growth. Various environmental and physiological factors contribute to disturbed reproductive health status leading to infertility problems in mammalian species. In the last couple of decades a significant number of studies have been conducted to investigate the transcriptome of reproductive tissues and organs in relation to the various reproductive health issues including endometritis, polycystic ovarian syndrome (PCOS), intrauterine growth restriction (IUGR), preeclampsia, and various age-associated reproductive disorders. Among others, the post-transcriptional regulation of genes by small noncoding miRNAs contributes to the observed transcriptome dysregulation associated with reproductive pathophysiological conditions. MicroRNAs as a class of non-coding RNAs are also known to be involved in various pathophysiological conditions either in cellular cytoplasm or they can be released to the extracellular fluid via membrane-bounded extracellular vesicles and proteins. The present review summarizes the cellular and extracellular miRNAs and their association with the etiology of major reproductive pathologies including PCOS, endometritis, IUGR and age-associated disorders in various mammalian species.

Regulation of Nrf2 and NF-κB during lead toxicity in bovine granulosa cells.

Lead (Pb), one of the pervasive and protracted environmental heavy metals, is believed to affect the female reproductive system in many species. The Nrf2 and NF-κB are the two key transcriptional factors regulating cellular redox status and response against stress and inflammation respectively, showing an interaction between each other. The aim of this study is to investigate the effect of Pb on bovine granulosa cells (GCs) and its association with the regulation of Nrf2 and NF-κB pathways. For this, bovine GCs were cultured in vitro and exposed to different doses of Pb for 2 h. Cellular response to Pb insult was investigated 24 h post treatment. Results showed that exposure of GCs to Pb-induced ROS accumulation and protein carbonylation. Additionally, GCs exhibited reduction in cell viability and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). This was accompanied by cell cycle arrest at G0/G1 phase. Moreover, Pb downregulated both Nrf2 and NF-κB and their downstream genes. Lead increased the expression of endoplasmic reticulum (ER) stress marker genes (GRP78 and CHOP) and the proapoptotic gene (caspase-3) while the antiapoptotic gene (BCL-2) was reduced. Our findings suggest that Pb-driven oxidative stress affected GCs proliferation, enhances ER stress, induces cell cycle arrest and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk. However, further functional analysis is required to explain different aspects of Nrf2 and NF-κB interactions under metal challenge.

Quercetin supports bovine preimplantation embryo development under oxidative stress condition via activation of the Nrf2 signalling pathway.

Nrf2 is a master regulator for antioxidant machinery against oxidative stress in bovine preimplantation embryos. The endogenous or exogenous modulation of Nrf2-KEAP1 system in bovine embryos may contribute to the understanding of the mechanisms behind the response of embryos to stress conditions. Therefore, here we aimed to investigate the protective effect of quercetin on bovine preimplantation embryos exposed to higher atmospheric oxygen concentration. For that, blastocysts, which were developed from zygotes cultured in media supplemented with or without quercetin under high oxygen level (20%), were subjected intracellular ROS level and mitochondrial analysis, and determining blastocyst formation rate and total cell number. Moreover, mRNA and protein expression level of Nrf2 and selected downstream antioxidant genes were investigated in the resulting blastocysts. Quercetin supplementation in vitro culture did not affect cleavage and blastocyst rate until day 7. However, quercetin supplementation resulted in higher blastocyst total cell number and reduction of intracellular ROS level accompanied by increasing mitochondrial activity compared with control group in both day 7 and day 8 blastocysts. Moreover, quercetin supplementation induced mRNA and protein of Nrf2 with subsequent increase in the expression of downstream antioxidants namely: NQO1, PRDX1, CAT and SOD1 antioxidants. In conclusion, quercetin protects preimplantation embryos against oxidative stress and improves embryo viability through modulation of the Nrf2 signalling pathway.

The Role of MicroRNAs in Mammalian Fertility: From Gametogenesis to Embryo Implantation.

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.

Sexual dimorphic expression and release of transcription factors in bovine embryos exposed to oxidative stress.

Sexually dimorphic differences in genome activity, which is orchestrated by transcription factors (TFs), could explain the differential response of male and female embryos to environmental stressors. To proof this hypothesis, the expression of cellular and extracellular TFs was investigated in male and female bovine embryos in vitro cultured either under low (5%) or high (20%) oxygen levels. The intracellular reactive oxygen species (ROS), total cell number, expression of nuclear factor (erythroid-derived 2) factor 2 (NFE2L2), Krüppel-like factor 4 (KLF4), notch receptor 1 (NOTCH1), E2F transcription factor 1 (E2F1), and SREBF2 along with extracellular vesicles (EVs) biogenesis genes were assessed at the blastocyst stage and their released EVs. Low blastocyst rate in both sexes due to oxidative stress (OS) was accompanied by increased ROS accumulation and reduced cell number in female embryos. The messenger RNA and protein levels of NFE2L2, as well as KLF4 expression, were higher in male embryos exposed to OS compared with female embryos. However, the expression of NOTCH1 and E2F1 was higher in female embryos cultured in high oxygen level. Male embryos exposed to OS released more EVs enriched with NFE2L2, superoxide dismutase 1, and NOTCH1 accompanied by elevated expression of EVs biogenesis genes. Accordingly, differential expression of TFs and their release into spent media could partially explain the sexual dimorphic response of bovine embryos to environmental stresses.

Endogenous and Exogenous Modulation of Nrf2 Mediated Oxidative Stress Response in Bovine Granulosa Cells: Potential Implication for Ovarian Function.

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.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

BACKGROUND: Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. RESULTS: The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. CONCLUSION: In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

MicroRNAs: tiny molecules with a significant role in mammalian follicular and oocyte development.

The genetic regulation of female fertility (follicular development, oocyte maturation and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth and embryo development. These studies have applied a variety of approaches, including conditional knockout of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation.

Clinical and subclinical endometritis induced alterations in bovine endometrial transcriptome and miRNome profile.

BACKGROUND: 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). RESULT: 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. CONCLUSION: 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.

MicroRNA-183-96-182 Cluster Regulates Bovine Granulosa Cell Proliferation and Cell Cycle Transition by Coordinately Targeting FOXO1.

Large-scale expression profiling of micro-RNAs (miRNAs) in bovine granulosa cells from dominant and subordinate follicles on Day 19 of the estrous cycle revealed enriched micro-RNA-183-96-182 cluster miRNAs in preovulatory dominant follicles that coordinately regulate the forkhead box protein O1 (FOXO1) gene. However, little is known about the role of this cluster in bovine granulosa cell function. We used an in vitro granulosa cell culture model to investigate this role. Granulosa cells aspirated from small growing follicles (3-5 mm in diameter) were cultured in Dulbecco modified Eagle medium/F-12 medium supplemented with fetal bovine serum and transfected with locked nucleic acid-based miRNA mimics, inhibitors, and corresponding negative controls. Overexpression of the miRNA cluster resulted in suppression of FOXO1 mRNA and protein, whereas inhibition of the cluster increased expression of FOXO1 mRNA. Overexpression also increased the relative rate of cell proliferation, whereas inhibition slowed it down. Similarly, the proportion of cells under G0/G1 arrest declined, whereas the ratio of cells in S phase increased in response to miR-183-96-182 overexpression. Selective knockdown of FOXO1 mRNA using anti-FOXO1 small interfering RNA increased the rate of granulosa cell proliferation, decreased the proportion of cells under G0/G1 arrest, and increased the proportion of cells in the S phase of cell cycle. Our data suggest that miR-183-96-182 cluster miRNAs promote proliferation and G1/S transition of bovine granulosa cells by coordinately targeting FOXO1, suggesting a critical role in granulosa cell function. MicroRNA-183-96-182 cluster regulates bovine granulosa cell function by targeting FOXO1 gene.

Potential role of microRNAs in mammalian female fertility.

Since the first evidence for the involvement of microRNAs (miRNAs) in various reproductive processes through conditional knockout of DICER, several studies have been conducted to investigate the expression pattern and role of miRNAs in ovarian follicular development, oocyte maturation, embryo development, embryo-maternal communication, pregnancy establishment and various reproductive diseases. Although advances in sequencing technology have fuelled miRNA studies in mammalian species, the presence of extracellular miRNAs in various biological fluids, including follicular fluid, blood plasma, urine and milk among others, has opened a new door in miRNA research for their use as diagnostic markers. This review presents data related to the identification and expression analysis of cellular miRNA in mammalian female fertility associated with ovarian folliculogenesis, oocyte maturation, preimplantation embryo development and embryo implantation. In addition, the relevance of miRNAs to female reproductive disorders, including polycystic ovary syndrome (PCOS), endometritis and abnormal pregnancies, is discussed for various mammalian species. Most importantly, the mechanism of release and the role of extracellular miRNAs in cell-cell communication and their potential role as non-invasive markers in female fertility are discussed in detail. Understanding this layer of regulation in female reproduction processes will pave the way to understanding the genetic regulation of female fertility in mammalian species.

Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos.

Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes' thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability.

Endometrial DNA methylation signatures during the time of breeding in relation to the pregnancy outcome in postpartum dairy cows fed a control diet or supplemented with rumen-protected methionine.

Post calving metabolic stress reduces the fertility of high producing dairy cows possibly by altering the expression of genes in the maternal environment via epigenetic modifications. Therefore, this study was conducted to identify endometrial DNA methylation marks that can be associated with pregnancy outcomes in postpartum cows at the time of breeding. For this, twelve days post-calving, cows were either offered a control diet or supplemented daily with rumen-protected methionine. Cows showing heat 50-64 days postpartum were artificially inseminated. Endometrial cytobrush samples were collected 4-8 h after artificial insemination and classified based on the pregnancy out comes as those derived from cows that resulted in pregnancy or resulted in no pregnancy. The DNAs isolated from endometrial samples were then subject to reduced representative bisulfite sequencing for DNA methylation analysis. Results showed that in the control diet group, 1,958 differentially methylated CpG sites (DMCGs) were identified between cows that resulted in pregnancy and those that resulted in no pregnancy of which 890 DMCGs were located on chr 27: 6217254-6225600 bp. A total of 537 DMCGs were overlapped with 313 annotated genes that were involved in various pathways including signal transduction, signalling by GPCR, aldosterone synthesis and secretion. Likewise, in methionine supplemented group, 3,430 CpG sites were differentially methylated between the two cow groups of which 18.7% were located on Chr27: 6217254-6225600 bp. A total of 1,781 DMCGS were overlapped with 890 genes which involved in developmental and signalling related pathways including WNT-signalling, focal adhesion and ECM receptor interaction. Interestingly, 149 genes involved in signal transduction, axon guidance and non-integrin membrane-ECM interactions were differentially methylated between the two cow groups irrespective of their feeding regime, while 453 genes involved in axon guidance, notch signalling and collagen formation were differentially methylated between cows that received rumen protected methionine and control diet irrespective of their fertility status. Overall, this study indicated that postpartum cows that could potentially become pregnant could be distinguishable based on their endometrial DNA methylation patterns at the time of breeding.

Extracellular Vesicles as Mediators of Environmental and Metabolic Stress Coping Mechanisms During Mammalian Follicular Development.

Extracellular vesicles are evolutionarily conserved nano-sized phospholipid membraned structures and released from virtually all types of cells into the extracellular space. Their ability to carry various molecular cargos (mRNA, miRNA, proteins, and lipids) from one cell to the other to exert functional impact on the target cells enables them to play a significant role in cell to cell communication during follicular development. As the molecular signals carried by extracellular vesicles reflect the physiological status of the cells of origin, they are expected to mediate any effect of environmental or metabolic stress on the follicualr cells and the growing oocyte. Recent studies have evidenced that reproductive cells exposed to various environmental stressors (heat and oxidative stress) released extracellular vesicles enriched with mRNA and miRNA associated with stress response mechanisms. Moreover, the metabolic status of post-calving cows could be well-reflected in the follicular extracellular vesicle's miRNA profile, which signified the potential role of extracellular cellular vesicle molecular signals in mediating the effect of metabolic stress on follicular and oocyte development. In the present review, the potential role of extracellular vesicles in mediating the effect of environmental and metabolic stress in various reproductive cells and oocytes are thoroughly discussed Moreover, considering the importance of extracellular vesicles in shuttling protective or rescuing molecular signals during stress, their potential usage as means of targeted delivery of molecules to mitigate the effect of stress on oocytes are addressed as the focus of future research.