Heat stress negatively affects reproduction in cattle by disrupting the normal function of ovarian granulosa cells (GCs), ultimately leading to oxidative damage and cell death via apoptosis. Heme oxygenase-1(HO-1) is a member of the heat shock protein family, which are associated with cellular antioxidant defenses and anti-apoptotic functions. Recent studies demonstrated that HO-1 is upregulated in heat-stressed cells. In the present study, we investigated the expression of HO-1 in bovine GCs transiently exposed to heat stress and characterized the expression and activity of key oxidative stress enzymes and molecules. We show that heat stress induced oxidative stress and apoptosis, and enhanced Nrf2 and HO-1 expression in primary GC cultures. Knocking down HO-1 expression using siRNA exacerbated both oxidative stress and apoptosis, whereas pre-treating GCs with hemin, which induces HO-1 expression, partially prevented these effects. These findings demonstrate that HO-1 attenuates heat stress-induced apoptosis in bovine GCs by decreasing production of reactive oxygen species and activating the antioxidant response.
Non-esterified fatty acid (NEFA), one of negative energy balance (NEB)'s most well-known products, has a significant impact on cows' reproductive potential. Our study used an in vitro model to investigate the deleterious effects of NEFA on bovine granulosa cells (BGCs) and its underlying molecular mechanism. The results showed that high levels of NEFA led to the accumulation of reactive oxygen species (ROS), increased the expression of apoptosis-related factors such as Bcl2-Associated X/B-cell lymphoma-2 (Bax/Bcl-2) and Caspase-3, and down-regulated steroid synthesis-related genes such as sterol regulatory element binding protein 1 (SREBP-1), cytochrome P450c17 (CYP17), and cytochrome P450 aromatase (CYP19), to promote oxidative stress, cell apoptosis, and steroid hormone synthesis disorders in BGCs. In addition, NEFA significantly inhibited phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (p-AKT) activity and increased forkhead box O1 (FoxO1) expression. To further explore the role of the PI3K/AKT/FoxO1 signaling pathway in NEFA, we found that pretreatment with AKT-specific activator SC79 (5 mg/mL) for 2 h or transfection with FoxO1 knockdown siRNA in BGCs could alleviate the negative effects of NEFA treatment by decreasing Bax/Bcl-2 ratio and Caspase-3 expression, and upregulating SREBP-1, CYP17, and CYP19 expression. Meanwhile, SC79 significantly inhibited NEFA-induced dephosphorylation and massive nuclear translocation of FoxO1. Taken together, the NEFA induced oxidative stress, apoptosis, and steroid hormone synthesis disorders in BGCs by inhibiting the PI3K/AKT pathway that regulates FoxO1 phosphorylation and nuclear translocation. Our findings help to clarify the molecular mechanisms underlying the negative effects of high levels of NEFA on BGCs.
The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.
Oocyte maturation is a dynamic process characterized by a lack of oocyte transcriptional activity following the breakdown of the germinal vesicle during the progression from prophase I until metaphase II arrest. During follicular development, intercellular communication between the oocyte and the surrounding cumulus cells can influence oocyte maturation and oocyte quality. One potential mechanism for molecule transfer into and between cells of the cumulus oocyte complex (COC) is through microvesicles found in follicular fluid. The objective of this project was to identify the expression of a specific miRNA (MIR21) in microvesicles isolated from porcine follicular fluid and to determine their impact on in vitro oocyte maturation in media lacking follicle stimulating hormone (FSH) and luteinizing hormone (LH). Microvesicles were isolated from follicular fluid of large (8-12 mm) follicles from ovaries collected from a local abattoir. Following ultracentrifugation of follicular fluid, microvesicles were utilized for total RNA extraction or were resuspended in in vitro maturation media with or without gonadotropins. Total RNA was utilized to conduct quantitative RT-PCR for MIR21. In vitro maturation conditions included: control maturation media (MM) with LH and FSH, MM media without either LH and FSH or microvesicles, MM without LH and FSH but with microvesicles, and MM with LH and FSH and microvesicles, (n= 4 replications per treatment). Utilization of stem-loop quantitative RT-PCR demonstrated the presence of MIR21 in total RNA extracted from microvesicles. Following in vitro maturation, the percentage of oocytes achieving MII arrest in the control treatment was 64.5 ± 2.8%. Removal of both LH and FSH reduced maturation rate (37.2 ± 4.0%, P < 0.001) compared to control conditions. Interestingly, addition of microvesicles increased maturation rate (52.2 ± 2.9%; P < 0.01) when added to MM lacking LH and FSH despite being significantly less than control conditions (P < 0.03). Addition of microvesicles to MM containing FSH and LH did not improve maturation rate (65.5 ± 5.9%) compared to control conditions. Collectively, these data demonstrate the ability of follicular fluid microvesicles containing miRNA to improve in vitro maturation of porcine oocytes in the absence of gonadotropins. This project was supported by National Research Initiative Competitive Grant no. 2008-35205-05309 and 2008-35205-18712 from the USDA National Institute of Food and Agriculture.
This paper described the extraction procedure and determination method for the total flavonoids in ethanol extract from the leaves of Abutilon theophrasti Medic., and evaluated antibacterial and antioxidant activity. Maximum extraction yield was achieved using 60% ethanol, 1 : 30 (g/mL) of a ratio of material to solvent, 20 min of extraction time, 40 kHz of ultrasonic frequency, 100 W of ultrasonic power, 600C of extraction temperature and two extraction cycles. Total flavonoids content was 16.79 RE mg/g medicinal materials. The extracts had effective antibacterial activity against 24 test strains from S. aureus and E. coli, MICs ranged from 2.18 to 8.7 mg/mL; it was also revealed that the extracts demonstrated high flavonids content and potent antioxidant activity, achieved by hydroxyl radical, DPPH radical and ABTS radical scavenging. These results indicated thathe extract may be a promising plant demonstrating antibacterial and antioxidant activities.
Transgenic technology has been applied mainly in the study of gene structure and function in model organisms and gene therapy for human diseases. Transgenic technology has potential for rapidly improving quantity and quality of agricultural products, compared to traditional selection and breeding methods in domestic animals that are time consuming when attempting to alter the desired allele frequency for specific traits. Additionally, transgenic animals can be used as biomedical research models or directly for human health, by producing recombinant pharmaceutical proteins and/or organs for xenotransplantation. Due to the advantage of bypassing the need of embryonic stem (ES) cells that are difficult to isolate in domestic animal species, cell-based method of transgenesis followed by somatic cell nuclear transfer (SCNT) is currently widely applied. However, due to the limitations in making genetic modifications and SCNT, producing genetically modified animals is still inefficient. Fortunately, the current advancement of new techniques and methods in both gene targeting (Urnov et al., 2010) and abilities to produce pluripotent stem cells (Voigt and Serikawa, 2009) holds great promises for this field. In this chapter, we will review the recent progress and technical route of the cell-based method of transgenesis by SCNT and discuss the newly emerging methods to enrich the gene targeting frequency of somatic cells. We will also discuss factors to improve the efficiency of SCNT and our future perspectives on the promises of this field.
Abstract The faithful execution of molecular programme underlying oocyte maturation and meiosis is vital to generate competent haploid gametes for efficient mammalian reproduction. However, the organization and principle of molecular circuits and modules for oocyte meiosis remain obscure. Here, we employed the recently developed single‐cell RNA‐seq technique to profile the transcriptomes of germinal vesicle (GV) and metaphase II (MII) oocytes, aiming to discover the dynamic changes of mRNAs and long non‐coding RNAs (lncRNAs) during oocyte in vitro meiotic maturation. During the transition from GV to MII, total number of detected RNAs (mRNAs and lncRNAs) in oocytes decreased. Moreover, 1,807 (602 up‐ and 1,205 down‐regulated) mRNAs and 313 (177 up‐ and 136 down‐regulated) lncRNAs were significantly differentially expressed (DE), i.e., more mRNAs down‐regulated, but more lncRNAs up‐regulated. During maturation of pig oocytes, mitochondrial mRNAs were actively transcribed, eight of which (ND6, ND5, CYTB, ND1, ND2, COX1, COX2 and COX3) were significantly up‐regulated. Both DE mRNAs and targets of DE lncRNAs were enriched in multiple biological and signal pathways potentially associated with oocyte meiosis. Highly abundantly expressed mRNAs (including DNMT1, UHRF2, PCNA, ARMC1, BTG4, ASNS and SEP11) and lncRNAs were also discovered. Weighted gene co‐expression network analysis (WGCNA) revealed 20 hub mRNAs in three modules to be important for oocyte meiosis and maturation. Taken together, our findings provide insights and resources for further functional investigation of mRNAs/lncRNAs in in vitro meiotic maturation of pig oocytes.
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