TY - JOUR
AU - Spicer, Leon, J
AB - Abstract Ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) is a multi-domain nuclear protein that plays an important role in epigenetics and tumorigenesis, but its role in normal ovarian follicle development remains unknown. Thus, the present study evaluated if UHRF1 mRNA abundance in bovine follicular cells is developmentally and hormonally regulated, and if changes in UHRF1 are associated with changes in DNA methylation in follicular cells. Abundance of UHRF1 mRNA was greater in granulosa cells (GC) and theca cells (TC) from small (<6 mm) than large (≥8 mm) follicles and was greater in small-follicle GC than TC. In GC and TC, fibroblast growth factor 9 (FGF9) treatment increased (P < 0.05) UHRF1 expression by 2-fold. Also, luteinizing hormone (LH) and insulin-like growth factor 1 (IGF1) increased (P < 0.05) UHRF1 expression in TC by 2-fold, and forskolin (an adenylate cyclase inducer) alone or combined with IGF1 increased (P < 0.05) UHRF1 expression by 3-fold. An E2F transcription factor inhibitor (E2Fi) decreased (P < 0.05) UHRF1 expression by 44% in TC and by 99% in GC. Estradiol, progesterone, and dibutyryl-cAMP decreased (P < 0.05) UHRF1 mRNA abundance in GC. Treatment of GC with follicle-stimulating hormone (FSH) alone had no effect but when combined with IGF1 enhanced the UHRF1 mRNA abundance by 2.7-fold. Beauvericin (a mycotoxin) completely inhibited the FSH plus IGF1-induced UHRF1 expression in small-follicle GC. Treatments that increased UHRF1 mRNA (i.e., FGF9) in GC tended to decrease (by 63%; P < 0.10) global DNA methylation, and those that decreased UHRF1 mRNA (i.e., E2Fi) in GC tended to increase (by 2.4-fold; P < 0.10) global DNA methylation. Collectively, these results suggest that UHRF1 expression in both GC and TC is developmentally and hormonally regulated, and that UHRF1 may play a role in follicular growth and development as well as be involved in ovarian epigenetic processes. Introduction Ubiquitin-like with plant homeodomain and really interesting new gene (RING) finger domains 1 (UHRF1) gene encodes a member of a subfamily of RING-finger type E3 ubiquitin ligases (Bostick et al., 2007; Ge et al., 2016; Jia et al., 2016). The resultant UHRF1 protein binds to specific DNA sequences and recruits DNA methyltransferase 1 (DNMT1) (Sharif et al., 2007; Rothbart et al., 2012; Liu et al., 2013), playing a major role in the G1/S transition stage of the cell cycle by regulating gene expression (Nishiyama et al., 2013; Qin et al., 2015; Jia et al., 2016). In particular, UHRF1 specifically recognizes hemimethylated DNA (i.e., a single CpG that is methylated on one strand) during DNA replication and recruits DNMT1 to add a methyl group to the unmethylated strand (Arita et al., 2008), the latter of which is important for maintaining DNA methylation patterns in daughter cells after mitosis (Bostick et al., 2007). Moreover, UHRF1 is regarded as a hub protein (i.e., a highly connected protein in a protein–protein interaction network) for the integration of epigenetic information (Arima et al., 2004; Jenkins et al., 2005; Ge et al., 2016; Zhao et al., 2017). Recent studies have shown that UHRF1 may act as an oncogenic factor with the potential to cause cancer development and progression. In fact, UHRF1 upregulation has been reported in various tumors, such as ovarian (Yan et al., 2015a, 2015b), breast (Geng et al., 2013), prostate (Babbio et al., 2012; Jazirehi et al., 2012), colorectal (Kofunato et al., 2012), and bladder (Unoki et al., 2009). Because UHRF1 is associated with poor prognosis and clinical outcomes (Liang et al., 2015b; Jia et al., 2016; Zhang et al., 2016), UHRF1 is considered a potential novel diagnostic marker of cancer and prognostic factor (Alhosin et al., 2016; Ge et al., 2016). Also, UHRF1 is considered to be a key epigenetic regulator playing a role in DNA maintenance and methylation (Jin et al., 2010; Tien et al., 2011; Zhao et al., 2017) and regulates proliferation of non-ovarian cells (Liu et al., 2020). For example, UHRF1 knockdown using small-interfering RNA (siRNA) increased the global DNA methylation levels and overexpression caused global DNA hypomethylation in esophageal squamous cell carcinoma cells (Nakamura et al., 2016). As mentioned earlier, UHRF1 is involved in adding methyl groups to DNA via recruitment of DNMT1, which then methylates DNA, and so why UHRF1 overexpression causes decreased global DNA methylation is likely because excess UHRF1 causes destabilization (Sharif et al., 2007) and degradation (Qin et al., 2015; Nakamura et al., 2016) of DNMT1, thereby reducing DNA methylation. Exposure to environmental contaminants of anthropological (e.g., pesticides) or natural (e.g., mycotoxins) origin are associated with adverse effects on and impairment of several physiological systems, including disruption of reproductive function (Cortinovis et al., 2013; Guillette et al., 2016; Bertero et al., 2018). Previous studies conducted in the authors’ lab have shown that exposure to endocrine disruptor molecules such as glyphosate (GLYP), fumonisin B1 (FB1), and beauvericin (BEA) might lead to reproductive disorders through the impairment of ovarian cell proliferation and steroidogenesis (Cortinovis et al., 2014; Albonico et al., 2016, 2017; Perego et al., 2017). Environmental toxins are also thought to alter epigenetic genes in vertebrates (Guillette et al., 2016; Huang et al., 2019), and recent evidence indicates that DNA methylation is a candidate mechanism for environmental effects on gene transcription (Lou et al., 2014; Houtepen et al., 2016). The disruption of an epigenetic regulator such as UHRF1 within ovarian follicles by environmental factors could impact long-term generational effects if oocyte (OC) gene expression (e.g., maternally inherited genes) is altered. Perhaps, the exposure to these molecules might have long-term generational effects through the control of epigenetic regulators such as UHRF1 and that aberrant intrafollicular UHRF1 production might lead to ovarian disorders. In microarray studies, Shi and Zhang (2017) working with epithelial ovarian cancer cells and Schütz et al. (2018) working with fibroblast growth factor 9 (FGF9)-treated bovine theca cells (TC) discovered that UHRF1 expression was upregulated. Also, FGF9 and other members of FGF family have a significant impact on ovarian function, including angiogenesis and granulosa cells (GC) proliferation and steroidogenesis (Chaves et al., 2012; Schreiber and Spicer, 2012; Price, 2016) as does IGF1 (Spicer and Echternkamp, 1995; Webb and Campbell, 2007) and EGF (Driancourt and Thuel, 1998; Richani and Gilchrist, 2018). The two main steroidogenic cell layers of the ovarian follicle, GC and TC, responding to follicle-stimulating hormone (FSH) and luteinizing hormone (LH), respectively, work together to ultimately produce estradiol and induce maturation of OCs (Hsueh et al., 1984,, 2015). In cattle, FGF9 regulates ovarian function by acting as an anti-differentiation factor, increasing TC and GC proliferation via induction of cell cycle proteins (Totty et al., 2017), and decreasing steroidogenesis in both GC (Schreiber and Spicer, 2012) and TC (Schreiber et al., 2012) by downregulating the expression of enzymes involved in steroidogenesis and through the inhibition of cyclic adenosine monophosphate (cAMP) signal transduction. To the authors’ knowledge, the developmental and hormonal regulation of UHRF1 gene expression in bovine GC and TC has not yet been investigated. Thus, the aim of the present study was to determine if UHRF1 expression in follicular cells changes during follicular growth and if UHRF1 mRNA is regulated by steroidogenic hormones and/or various growth factors. It was hypothesized that UHRF1 gene expression would be hormonally responsive and change during follicular development. Secondary objectives were to determine if environmental contaminants change UHRF1 mRNA levels, and if changes in UHRF1 gene expression were associated with functional (i.e., global DNA methylation) changes in GC. Materials and Methods Tissue, reagents, and hormones Ovaries from beef heifers were collected at a slaughterhouse where humane slaughter practices were followed, according to the US Department of Agriculture guidelines. The reagents used in cell culture were: Ham’s F-12 (F12), Dulbecco modified Eagle medium (DMEM), gentamicin, sodium bicarbonate, trypan blue, collagenase, deoxyribonuclease (DNase), dihydrotestosterone (DHT), progesterone (P4), estradiol (E2), androstenedione (A4), forskolin (Fsk), dibutyryl cAMP (dbcAMP), GLYP (PESTANAL), FB1, and BEA from Sigma-Aldrich Chemical Company (St. Louis, MO), and fetal calf serum (FCS) from Atlanta Biologicals (Flowery Branch, GA). The reagents used in RNA extraction were: TRI Reagent Solution and diethyl pyrocarbonate (DEPC)-treated water from Life Technologies, Inc. (Gaithersburg, MD), and isopropyl alcohol and ethanol from Pharmco Products Inc. (Brookfield, CT). The hormones and growth factors used in cell culture were ovine FSH (FSH activity: 15 × NIH-FSH-S1 U/mg) and LH (NIDDK-oLH-26, activity 1.0 × NIH-LH-S1 U/mg) from the National Hormone and Pituitary Program (Torrance, CA); carrier-free recombinant human insulin-like growth factor (IGF)-1, FGF9, and epidermal growth factor (EGF) from R&D Systems (Minneapolis, MN); testosterone from Steraloids (Wilton, NH); and E2F inhibitor (E2Fi; HLM006474) from EMD Millipore Corp. (Billerica, MA) shown to inhibit E2F1, E2F2, E2F3, and E2F4 transcription factors (Ma et al., 2008; Kurtyka et al., 2014). These transcription factors are involved in cell cycle progression and regulate cell proliferation, apoptosis, and differentiation (Qin et al., 2006; Thurlings and de Bruin, 2016; Morrell et al., 2020). Cell culture Ovaries from nonpregnant beef heifers were collected in various batches from a slaughterhouse, GC and TC isolated, and resuspended in medium (1:1 DMEM and F12, glutamine, sodium bicarbonate, and gentamicin) containing collagenase and DNase as previously described (Schrieber and Spicer, 2012; Zhang et al., 2017). Viability of GC from small (1 to 5 mm) and large (8 to 20 mm) follicles and of TC from large follicles was determined by trypan blue exclusion method (Adashi et al., 1987) which averaged 64%, 55%, and 95%, respectively. The purity of TC prepared this way was >90% (Roberts and Skinner, 1990; Spicer and Stewart, 1996; Spicer et al., 2008). On average, 2.0 × 105 viable cells were plated per well on 24-well Falcon multiwell plates (No. 3047; Becton Dickinson, Lincoln Park, NJ) in 1 mL of medium containing 10% FCS. Plates were maintained in a humidified 95% air and 5% CO2 environment at 38.5 °C changing medium every 24 h. Cells were cultured in the presence of 10% FCS for the first 48 h of culture to procure an optimal attachment (Zhang et al., 2017). After 48 h, cells were washed twice with serum-free medium, and the different treatments were applied in serum-free medium. At the end of the treatment period, medium was aspirated from each well and cells from two replicate wells were lysed in 0.5 mL of TRI reagent solution, and RNA was isolated from cell lysates as described previously (Spicer and Aad, 2007; Spicer et al., 2008; Zhang et al., 2017). Concentrations of RNA were assessed with a spectrophotometer at 260 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE), and RNA was then diluted to 10 ng/μL in DEPC-treated water. Real-time PCR analysis The UHRF1 (3,933 bp, accession no. NM_001103098) forward primer was constructed between 3,642 and 3,669 bp with a Tm of 54.1 °C and had a sequence of 5′-GGGAATGATTCGTTAATGTTTCTAACTT-3′. The reverse UHRF1 primer was constructed between 3,742 and 3,718 bp with a Tm of 57.0 °C and a sequence of 5′-CTCCAGTTTGTCTGC-AGTTATGTGA-3′. The UHRF1 probe was designed between 3,676 and 3,712 bp with a Tm of 61.9 °C with a sequence of 5′-AGTTTGCAGCTTATACCTCAACAGAACAGGGATATTT-3′. The primers and probe were purchased from Integrated DNA Technologies Inc. (San Diego, CA). The UHRF1 primers also spanned introns to reduce gDNA contamination (forward primer was located in exon 2, bases 3,061 to 3,082 [accession no. AC_000159], and reverse primer was located in exon 3, bases 4,865 to 4,882 of its gene; the TaqMan Probe was located in exon 2 bases 3,088 to 3,109). The probe possesses a fluorescein, a napthylene-azo modifier, and an 3’Iowa Black fluorescein quencher as the 5′ reporter, internal quencher, and the 3′ quencher dyes, respectively. A “highly similar sequences” BLAST query search (http://www.ncbi.nlm.nih.gov/BLAST) was conducted for the primers and probe to ensure their specificity. This was also done to assure that they were not designed from any homologous regions coding for other genes. Quantification of UHRF1 mRNA abundance was assessed using one-step quantitative with iTaqTM Universal Probe One-Step Kit (Bio-Rad, Hercules, CA), on a CFX96 Real-Time System in a 96-well plate (Bio-Rad, Hercules, CA). The total reaction volume of 20 µL consists of 200 nM of forward and reverse UHRF1 primers and 100 nM of UHRF1 probe. Samples were placed in duplicate wells on 96-well plates to determine an average threshold cycle (Ct) value. Target gene expression was normalized to constitutively expressed 18S rRNA (supplied as a VIC probe; TaqMan Ribosomal RNA Control Reagent, Applied Biosystems Inc.). The relative quantity of target gene mRNAs was expressed as 2−ΔΔCt using the relative comparative Ct method as previously described (Livak and Schmittgen, 2001; Voge et al., 2004; Aad et al., 2012; Zhang et al., 2017). The housekeeping gene, human 18S rRNA (accession no. X03205.1), was selected because previous studies show that it to be a stable gene over a variety of treatments (Schmittgen and Zakrajsek, 2000; Sekar et al., 2000; Tsuji et al., 2002; Voge et al., 2004; Zhang et al., 2017). Global DNA methylation level determination The total genomic DNA was extracted from GC, and the global DNA methylation levels were determined using the colorimetric enzyme-linked immunosorbent assay, MethylFlash Global DNA methylation (5-mC) Quantification kit (Epigentek Inc., Farmingdale, NY, USA), as previously described (Murphy et al., 2013; Oporto and Salazar, 2018) and according to the manufacturer’s instructions. This kit provides the levels of global DNA methylation in response to various treatments. Experimental design Experiment 1 was designed to determine if abundance of UHRF1 mRNA changes with follicle growth or differed between GC and TC in bovine ovarian follicles. Thus, UHRF1 mRNA abundance was evaluated in freshly collected GC and TC from small and large follicles. For large-follicle GC and TC, the samples were collected from individual follicles. For small-follicle GC and TC, each sample had pooled cells from 3 to 5 small follicles from individual ovaries. For exp. 1, each cell type had seven samples collected from at least three animals for both small and large follicles. These fresh cells were lysed in TRI reagent and extracted for RNA as described earlier. Experiment 2 was designed to evaluate the effect of FGF9 on UHRF1 mRNA abundance in TC from large follicles, given that these cells showed low expression of UHRF1 in exp. 1. Cells were cultured for 48 h in 10% FCS, washed twice with 0.5 mL of serum-free medium, and treated for 24 h in serum-free medium containing no additions (control) or two doses of FGF9 (30 or 100 ng/mL). After 24 h of treatment, medium was aspirated and cells were lysed in 0.5 mL of TRI reagent for RNA extraction as described earlier. Doses of FGF9 were based on previous studies (Schreiber et al., 2012; Totty et al., 2017; Zhang et al., 2017). Experiment 3 was designed to evaluate the effects of LH and IGF1, alone or combined, and Fsk (an inducer of adenylate cyclase), alone or combined with IGF1 on UHRF1 mRNA abundance in large-follicle TC. Cells were cultured as described for exp. 2 and the following seven treatments were applied for 24 h: Controls, LH (30 ng/mL) and IGF1 (30 ng/mL), alone or combined, and Fsk (4.1 µg/mL) alone or combined with IGF1. After a 24-h treatment, cellular RNA was collected as described earlier. Doses of IGF1, LH, and Fsk were based on previous studies (Spicer and Stewart, 1996; Spicer et al., 2002; Schreiber and Spicer, 2012). Experiment 4 was designed to test the effects of steroids, EGF, and E2Fi on UHRF1 mRNA abundance in TC from large follicles. Cells were cultured as described above and three treatments were applied for 24 h as follows: Controls, E2 (300 ng/mL), DHT (300 ng/mL), P4 (300 ng/mL), EGF (10 ng/mL), and E2Fi (50 µM). After a 24- h treatment, cellular RNA was collected as described earlier. Doses of steroids were based on previous studies (Spicer and Hammond, 1989; Zhang et al., 2017). Doses of EGF and E2Fi were also based on previous studies (Ma et al., 2008; Kurtyka et al., 2014; Zhang et al., 2017; Nichols et al., 2019). Experiment 5 was designed to evaluate the effects of A4, P4, dbcAMP, FGF9, and EGF on UHRF1 mRNA abundance in large-follicle GC, which like large-follicle TC expressed low levels of UHRF1 mRNA. Cells were cultured as previously described and the following six treatments were applied for 24 h: Controls, A4 (300 ng/mL), P4 (300 ng/mL), dbcAMP (0.25 mM), FGF9 (30 ng/mL), and EGF (30 ng/mL). After a 24-h treatment, cellular RNA was collected as described earlier. Doses of steroids, FGF9, EGF, and dbcAMP were based on previous studies (Spicer and Stewart, 1996; Spicer, 2005; Schreiber et al., 2012; Zhang et al., 2017). Experiment 6 was designed to evaluate the effect of E2 on UHRF1 mRNA abundance in large-follicle GC. Cells were cultured as previously described and treated with either no E2 (Controls) or E2 (300 ng/mL) for 24 h, and cellular RNA collected as described earlier. Dose of E2 was based on the previous studies (Spicer, 2005; Zhang et al., 2017; Nichols et al., 2019). Experiment 7 was designed to evaluate the effects of E2, A4, IGF1, E2Fi, and FGF9 on UHRF1 mRNA abundance in small-follicle GC, a cell type with high UHRF1 mRNA expression. Cells were cultured as previously described and the following six treatments were applied for 24 h: Controls, E2 (300 ng/mL), A4 (300 ng/mL), IGF1 (30 ng/mL), E2Fi (50 µM), and FGF9 (30 ng/mL). After a 24-h treatment, cellular RNA collected as described earlier. Doses of steroids, IGF1, FGF9, and E2Fi were based on exp. 4 and 5 and on previous studies (Spicer, 2005; Ma et al., 2008; Schreiber and Spicer, 2012; Zhang et al., 2017; Nichols et al., 2019). Experiment 8 was designed to test the effects of GLYP, BEA, and FB1, in the presence of FSH plus IGF1, on UHRF1 mRNA abundance in GC from small follicles. Cells were cultured as described for exp. 2 and the following six treatments were applied for 24 h: Controls, FSH (30 ng/mL), alone or combined with IGF1 (30 ng/mL), and either GLYP (30 µM), BEA (30 µM), or FB1 (30 µM) in the presence of FSH plus IGF1. After a 24-h treatment, cellular RNA collected as described earlier. Doses of FSH, IGF1, GLYP, BEA, and FB1 were based on previous studies (Spicer et al., 2002; Albonico et al., 2017; Perego et al., 2017; Zhang et al., 2017). Experiment 9 was designed to evaluate the effect of treatments that change UHRF1 mRNA expression, FGF9 (30 ng/mL), and E2Fi (50 µM), on cellular DNA methylation in small-follicle GC. The MethylFlash Global DNA methylation (5-mC) Quantification kit (Epigentek Inc.) was used to determine the global DNA methylation levels. Cells were cultured for 48 h in 10% FCS, washed, and treated for 24 h with either FGF9 or E2Fi. Then, medium was aspirated and cellular DNA collected as per the manufacturer’s instructions. Doses of FGF9 and E2Fi were based on exp. 7 and on previous studies (Ma et al., 2008; Schreiber and Spicer, 2012; Nichols et al., 2019). Statistical analyses Experiment 1 was analyzed as 2 × 2 factorial ANOVA with follicle size, cell type, and their interactions. Data from exp. 2 to 9 were analyzed as a 1-way ANOVA. For exp. 2 to 9, three different pools for each cell type were used as experimental/biological replicates with duplicate determinations for each pool (n = 6). Each pool of small-follicle GC (exp. 7, 8, and 9) was generated from a total volume of 6 to 8 mL of follicular fluid (from 25 to 30 ovaries) per pool within each experimental replicate. Each pool of large-follicle GC and TC (exp. 2 to 6) was obtained from five to seven follicles from three to five cows. The medium was applied to four wells and duplicate samples for each pool of cells were derived by combining RNA from two wells. Treatment effects and interactions were assessed using the general linear models procedure of the SAS for Windows (version 9.4, SAS Institute Inc., Cary, NC). Mean differences were determined by Fisher’s protected least significant differences test (Ott, 1977), if significant main effects in the ANOVA were detected. Data were presented as least square means ± SEM. Significance was determined at P < 0.05, and tendencies were declared at 0.05 < P < 0.10. Results Experiment 1: evaluation of mRNA abundance of UHRF1 in bovine ovarian follicles according to cell type and follicle size Abundance of UHRF1 mRNA was greater in small follicles (P < 0.05) for both GC (6.4-fold greater) and TC (3.0-fold greater) than large follicles (Figure 1). Abundance of UHRF1 was 2.3-fold greater in GC than in TC from small follicles (P < 0.05) but did not significantly differ between cell types in large follicles (P > 0.10) (Figure 1). Figure 1. Open in new tabDownload slide Levels of UHRF1 mRNA in freshly collected GC and TC from small and large follicles (exp. 1). Real-time PCR quantified steady-state mRNA levels were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of large follicle TC value. a–cMeans (± SEM; n = 7) without a common letter differ (P < 0.05). Figure 1. Open in new tabDownload slide Levels of UHRF1 mRNA in freshly collected GC and TC from small and large follicles (exp. 1). Real-time PCR quantified steady-state mRNA levels were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of large follicle TC value. a–cMeans (± SEM; n = 7) without a common letter differ (P < 0.05). Experiment 2: effect of FGF9 on UHRF1 mRNA abundance in large-follicle TC Doses of FGF9 affected (P < 0.01) UHRF1 mRNA abundance (Figure 2). Specifically, 30 ng/mL of FGF9 increased (P < 0.05) UHRF1 expression by 2.0-fold and 100 ng/mL of FGF9 increased (P < 0.05) UHRF1 expression by 2.6-fold (Figure 2). Figure 2. Open in new tabDownload slide In vitro effect of FGF9 (0, 30, or 100 ng/mL) on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 2). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Figure 2. Open in new tabDownload slide In vitro effect of FGF9 (0, 30, or 100 ng/mL) on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 2). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Experiment 3: effect of LH, IGF1, and Fsk on UHRF1 mRNA abundance in large-follicle TC Neither LH nor IGF1 affected (P > 0.10) UHRF1 mRNA abundance, while the combination of LH and IGF1 increased (P < 0.05) UHRF1 expression by 2.2-fold (Figure 3). Forskolin alone increased (P < 0.05) UHRF1 mRNA abundance by 2.8-fold; however, the addition of IGF1 did not statistically increase UHRF1 mRNA abundance above Fsk alone (3-fold increase). Figure 3. Open in new tabDownload slide In vitro effects of LH (30 ng/mL) and IGF1 (I; 30 ng/mL), alone or combined, and Fsk (4.1 μg/mL), alone or combined with IGF1, and E2Fi (50 μM) on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 3). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Figure 3. Open in new tabDownload slide In vitro effects of LH (30 ng/mL) and IGF1 (I; 30 ng/mL), alone or combined, and Fsk (4.1 μg/mL), alone or combined with IGF1, and E2Fi (50 μM) on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 3). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Experiment 4: effect of steroids, EGF, and E2Fi on UHRF1 mRNA abundance in large-follicle TC Treatment with P4, E2, DHT, or EGF had no effect (P > 0.10) on UHRF1 mRNA abundance in TC of large follicles (Figure 4). However, treatment with E2Fi decreased (P < 0.05) UHRF1 expression by 44% (Figure 4). Figure 4. Open in new tabDownload slide In vitro effects of steroids, EGF, and E2Fi on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 4). Treatments were applied for 24 h and were 300 ng/mL of P4, E2, or DHT, and 10 ng/mL of EGF and 50 µM of E2Fi. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and are expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Figure 4. Open in new tabDownload slide In vitro effects of steroids, EGF, and E2Fi on UHRF1 mRNA abundance in bovine TC from large follicles (exp. 4). Treatments were applied for 24 h and were 300 ng/mL of P4, E2, or DHT, and 10 ng/mL of EGF and 50 µM of E2Fi. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and are expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Experiment 5: effect of A4, P4, dbcAMP, FGF9, and EGF on UHRF1 mRNA abundance in large-follicle GC In large-follicle GC, FGF9 increased (P < 0.05) UHRF1 expression by 2-fold, whereas P4 and dbcAMP decreased (P < 0.05) UHRF1 mRNA abundance by 50.2% and 60.3%, respectively (Figure 5). In contrast, A4 and EGF had no effect (P > 0.10) on UHRF1 mRNA abundance in large-follicle GC (Figure 5). Figure 5. Open in new tabDownload slide In vitro effects of A4 (300 ng/mL), P4 (300 ng/mL), dbcAMP (0.25 mM), FGF9 (30 ng/mL), and EGF (30 ng/mL) on UHRF1 mRNA abundance in bovine GC from large follicles (exp. 5). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a–dMeans without a common letter differ (P < 0.05). Figure 5. Open in new tabDownload slide In vitro effects of A4 (300 ng/mL), P4 (300 ng/mL), dbcAMP (0.25 mM), FGF9 (30 ng/mL), and EGF (30 ng/mL) on UHRF1 mRNA abundance in bovine GC from large follicles (exp. 5). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a–dMeans without a common letter differ (P < 0.05). Experiment 6: effect of E2 on UHRF1 mRNA abundance in large-follicle GC In large-follicle GC, E2 decreased (P < 0.05) UHRF1 mRNA abundance by 51% from 1.0 ± 0.2 (Controls) to 0.49 ± 0.2 (E2 treated) relative abundance. Experiment 7: effect of E2, A4, IGF1, E2Fi, and FGF9 on UHRF1 mRNA abundance in small-follicle GC In small-follicle GC, E2 and E2Fi decreased (P < 0.05) UHRF1 mRNA abundance by 38.1% and 99.4%, respectively, whereas FGF9 enhanced (P < 0.05) UHRF1 expression by 1.7-fold (Figure 6). Treatment with A4 or IGF1 had no significant effect (P > 0.10) on UHRF1 expression (Figure 6). Figure 6. Open in new tabDownload slide In vitro effects of E2 (300 ng/mL), A4 (300 ng/mL), IGF1 (30 ng/mL), E2Fi (50 μM), and FGF9 (30 ng/mL) on UHRF1 mRNA abundance in bovine GC from small follicles (exp. 7). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a–dMeans without a common letter differ (P < 0.05). Figure 6. Open in new tabDownload slide In vitro effects of E2 (300 ng/mL), A4 (300 ng/mL), IGF1 (30 ng/mL), E2Fi (50 μM), and FGF9 (30 ng/mL) on UHRF1 mRNA abundance in bovine GC from small follicles (exp. 7). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a–dMeans without a common letter differ (P < 0.05). Experiment 8: effect of FSH, FSH plus IGF1, and of GLYP, BEA, and FB1 on UHRF1 mRNA abundance in small-follicle GC There was no significant effect of FSH on UHRF1 expression, but combined FSH plus IGF1 treatment increased (P < 0.05) UHRF1 mRNA abundance by 2.7-fold in small-follicle GC (Figure 7). GLYP and FB1 did not affect (P > 0.10) the stimulatory effect of FSH plus IGF1, while BEA completely inhibited (P < 0.05) FSH plus IGF1-induced UHRF1 mRNA abundance (Figure 7). Figure 7. Open in new tabDownload slide In vitro effects of GLYP (30 μM), BEA (30 μM), and FB1 (30 μM), in the presence of FSH (30 ng/mL) plus IGF1 (30 ng/mL), on UHRF1 mRNA abundance in bovine GC from small follicles (exp. 8). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Figure 7. Open in new tabDownload slide In vitro effects of GLYP (30 μM), BEA (30 μM), and FB1 (30 μM), in the presence of FSH (30 ng/mL) plus IGF1 (30 ng/mL), on UHRF1 mRNA abundance in bovine GC from small follicles (exp. 8). Treatments were applied in a serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a,bMeans without a common letter differ (P < 0.05). Experiment 9: effect of FGF9 and E2Fi on global cellular DNA methylation in small-follicle GC Treatment with FGF9 (30 ng/mL) tended to decrease (P < 0.10) methylation by 63%, whereas treatment with E2Fi (50 µM) tended to increase (P < 0.10) global cellular methylation in small-follicle GC by 2.4-fold (Figure 8). Figure 8. Open in new tabDownload slide In vitro effect of FGF9 (30 ng/mL) or E2Fi (50 µM) on global cellular methylation in bovine GC from small follicles (exp. 9). Treatments were applied in serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a–cMeans without a common letter tended to differ (P < 0.10). Figure 8. Open in new tabDownload slide In vitro effect of FGF9 (30 ng/mL) or E2Fi (50 µM) on global cellular methylation in bovine GC from small follicles (exp. 9). Treatments were applied in serum-free medium for 24 h. Real-time PCR quantified steady-state mRNA levels (± SEM; n = 6) were normalized to constitutively expressed 18S ribosomal RNA and expressed as a ratio of control values. a–cMeans without a common letter tended to differ (P < 0.10). Discussion According to the authors’ knowledge, the present study is the first to characterize changes in UHRF1 expression during follicular growth of any species and to show the hormonal regulation of UHRF1 mRNA within normal follicles. The results of the present study indicate that: 1) UHRF1 gene expression in GC and TC is developmentally and hormonally regulated; 2) FGF9 enhances UHRF1 mRNA expression in GC and TC; 3) E2Fi decreases UHRF1 mRNA abundance in GC and TC; 4) E2 and P4 decrease UHRF1 mRNA abundance in GC; 5) androgens have no effect on UHRF1 gene expression in GC or TC; 6) BEA decreases UHRF1 gene expression in GC from small follicles; and 7) treatments that increased and decreased UHRF1 mRNA tended to decrease and increase, respectively, global cellular methylation in GC. The presence of UHRF1 in the ovary has been previously reported in oncology studies. UHRF1 gene expression is upregulated and associated with cell proliferation and cancerous invasion in epithelial ovarian cancer in humans (Yan et al., 2015a, 2015b). Also, UHRF1 expression was reported to be greater in ovarian cancer cells compared with the expression of adjacent healthy tissues (Yan et al., 2015b; Shi and Zhang, 2017). A recent study conducted by Maenohara et al. (2017) showed that UHRF1 (protein) is localized in OCs and preimplantation embryos in mice, but they did not evaluate other ovarian cell types. The results of the present study show for the first time that UHRF1 mRNA is expressed and developmentally regulated in both GC and TC of cattle, and thus, UHRF1 may play a role in follicular growth and development. Specifically, UHRF1 expression was greater in GC and TC from small vs. large follicles, suggesting that hormones that drive follicular development/differentiation may be inhibiting UHRF1 gene expression, whereas hormones that promote cell proliferation and inhibit differentiation may be stimulating UHRF1 gene expression. Indeed, FGF9 known to be elevated in small follicles and inhibit differentiation (Schreiber et al., 2012; Schütz et al., 2016) stimulated UHRF1 mRNA expression in both TC and GC, whereas hormones that are known to be elevated in large follicles such as E2 and P4 (Stewart et al., 1996) inhibited UHRF1 mRNA expression in small-follicle GC (E2) and large-follicle GC (E2 and P4) (Figure 9). However, androgens (A4, DHT) had no significant effect on UHRF1 expression in TC or GC in the present study. Interestingly, Schütz et al. (2016) reported that E2 induces FGF9 mRNA in small-follicle GC but inhibits it in large-follicle TC, suggesting that the FGF9-UHRF1 response is dependent on the stage of follicle development/size (Figure 9). Consistent with the findings of the present study, UHRF1 is overexpressed in proliferative cells, whereas it is not expressed in differentiated tissues (Hopfner et al., 2000; Bronner et al., 2013). These and other researchers further speculate that UHRF1 overexpression in cancer cells helps maintain the cells in a proliferative state and prevent their differentiation (Bronner et al., 2007, 2013; Mulder et al., 2012). Whether UHRF1 is helping maintain an undifferentiated state in small-follicle GC and TC will require further elucidation. Figure 9. Open in new tabDownload slide Schematic model summarizing the hormonal regulation of UHRF1 production by GC and TC in small and large follicles. The hormones IGF1, FSH, and LH are stimulatory to E2 and A4 production. Increased free IGF1 in large follicles increases E2 production and decreases FGF9 mRNA in GC, and these changes cause a reduction in the UHRF1 production by GC and TC, which induces an overall increase in global DNA methylation by the dominant follicle. Figure 9. Open in new tabDownload slide Schematic model summarizing the hormonal regulation of UHRF1 production by GC and TC in small and large follicles. The hormones IGF1, FSH, and LH are stimulatory to E2 and A4 production. Increased free IGF1 in large follicles increases E2 production and decreases FGF9 mRNA in GC, and these changes cause a reduction in the UHRF1 production by GC and TC, which induces an overall increase in global DNA methylation by the dominant follicle. The FGF family consists of 23 members involved in several biological processes, such as cell proliferation, differentiation and migration, tissue repair, angiogenesis, and embryonic development (Ornitz et al., 1996; Drummond et al., 2007; Price, 2016). Each FGF preferentially binds to one or more of the four main FGF receptors (FGFR1-4) and their subtypes (Ornitz et al., 1996). The FGFRs that bind FGF9 (i.e., FGFR1-4) are expressed in both GC and TC of cattle (Parrott and Skinner, 1998; Berisha et al., 2004; Buratini et al., 2005), suggesting that FGF9 should affect GC and TC function in cattle. Indeed, FGF9 induced UHRF1 gene expression in both GC and TC in the present study. Similarly, FGF9 stimulates bovine GC and TC proliferation and inhibits bovine GC and TC steroidogenesis (Schreiber and Spicer, 2012; Schreiber et al., 2012; Totty et al., 2017). The presence of FGF9 mRNA in bovine follicular cells was first reported by Grado-Ahuir et al. (2011), who found it to be downregulated in GC of ovarian follicular cysts, which represent a major cause of impairment of reproductive success in dairy cattle. Therefore, it was hypothesized that decreased UHRF1 is required for normal follicle development and that any deviation in the normal changes in UHRF1 mRNA abundance might lead to abnormal follicular development. The present study showed that FGF9 treatment of GC increased UHRF1 mRNA and tended to decrease global cellular methylation, suggesting a functional link between UHRF1 expression and DNA methylation in GC. Similarly, overexpression of UHRF1 caused DNA hypomethylation in various carcinoma cells (Mudbhary et al., 2014; Nakamura et al., 2016). An explanation for UHRF1-induced DNA hypomethylation may be due to UHRF1 causing destabilization (Sharif et al., 2007) and degradation (Qin et al., 2015; Nakamura et al., 2016) of DNMT1, which is needed for DNA methylation. Interestingly, Zhang et al. (2014) using ovaries from an androgenized rat model to study polycystic ovarian syndrome (PCOS) and Xu et al. (2016) in GC from women with PCOS suggested that alterations in global and genome-wide DNA methylation may contribute to changes in gene expression and cause PCOS. As previously mentioned, UHRF1 expression is greater in ovarian cancer cells compared with the expression of the adjacent healthy tissues (Yan et al., 2015b; Shi and Zhang, 2017). Therefore, UHRF1 overexpression may also lead to abnormal follicular development, but further research will be required to verify this suggestion. The E2F transcription factor family is composed of 8 members (E2F1-8) encoding for 10 proteins involved in cell cycle progression, cell proliferation, and cell differentiation (Qi et al., 2015; Johnson et al., 2016; Wang et al., 2018). Specifically, E2F transcription factors form complexes with dimerization proteins, which regulate DNA replication and control the onset of S phase (Ertosun et al., 2016; Wang et al., 2018), and can upregulate UHRF1 transcription in human breast cells (Mousli et al., 2003; Unoki et al., 2004) and human lymphocytes (Abbady et al., 2005). In the present study, the inhibition of E2F transcription factors with E2Fi decreased UHRF1 mRNA abundance, suggesting that one or more of the eight E2F might be involved in the regulation of UHRF1 within the ovary. Interestingly, the inhibitory effect of E2Fi on UHRF1 expression was much greater in GC than TC. Other studies have shown that E2F regulates the function of human and rat GC (Putowski et al., 2001), mice GC (Yin et al., 2014), and bovine GC (Nichols et al., 2019; Morrell et al., 2020). Wang et al. (2018) recently showed that E2F transcription factors may also be involved in the response to DNA damage and play a role in DNA repair. According to their results, a DNA repair protein negatively regulates E2F transcription factors binding to their activation domain and inhibiting their transcriptional activities to arrest cell cycle progression allowing time for DNA repair (Wang et al., 2018). Previous studies have shown that UHRF1 also plays an active role in the DNA repair pathways (Liang et al., 2013, 2015a; Yang et al., 2013; Sidhu and Capalash, 2017). In the present study, E2Fi treatment of GC decreased UHRF1 mRNA and tended to increase global cellular methylation, further suggesting a functional link between UHRF1 expression and DNA methylation in GC. Similarly, UHRF1 knockdown using siRNA increased global DNA methylation levels in esophageal carcinoma cells (Nakamura et al., 2016). Further studies will be required to better understand the interaction between the various E2F and UHRF1 to promote DNA repair and proliferation in TC and GC. Additional studies are also needed to elucidate the regulation of UHRF1 expression in follicular cells and to better understand which of the eight E2F members play a role in UHRF1 regulation and determine the role and mechanism of action of UHRF1 during follicular development and ovarian epigenetic processes. Gonadotropins FSH and LH independently stimulate ovarian steroidogenesis via cAMP mediation (Weiss et al., 1976), but combined treatments of FSH plus IGF1 and LH plus IGF1 synergistically induce cAMP production in GC and TC, respectively, to levels much greater than any single treatment of FSH, LH, or IGF1 alone (Weiss et al., 1976; Jolly et al., 1997; Schreiber and Spicer, 2012). It is important to emphasize that IGF1 amplifies gonadotropin-induced cAMP via induction of FSH and LH receptors in GC (Minegishi et al., 2003; Spicer and Aad, 2007) and LH receptors in TC (Stewart et al., 1995; Huang et al., 2001). In the present study, dbcAMP, a cAMP analog (Schreiber et al., 2012; Robinson et al., 2018), inhibited UHRF1 mRNA in large-follicle GC, whereas a combined treatment with FSH plus IGF1 in small-follicle GC and LH plus IGF1 in large-follicle TC enhanced UHRF1 mRNA abundance. Similarly, Fsk, which promotes adenyl cyclase activity and enhances intracellular cAMP levels (Hedin and Rosberg, 1983; Meidan et al., 1992; Robinson et al., 2018), increased UHRF1 expression in TC of the present study. Thus, the particular effect of increased cAMP on UHRF1 gene expression may be cell-type and follicle-size dependent, stimulatory to small-follicle GC and large-follicle TC but inhibitory to large-follicle GC. Why these responses differ among cell types will require further elucidation. Nonetheless, the present studies support the idea that UHRF1 production decreases during follicular development and that factors driving this decrease as follicles enlarge include decreased FGF9 and increased E2, P4, and hormone-induced (e.g., IGF1 plus FSH or LH) cAMP production (Figure 9). As mentioned earlier, exposure to environmental contaminants of anthropological (e.g., pesticides and herbicides) or natural (e.g., mycotoxins) origin have been related with adverse effects on several physiological systems, including disruption of reproductive function (Cortinovis et al., 2013; Guillette et al., 2016; Bertero et al., 2018). Thus, another aim of the present study was to determine if exposure to possible endocrine disruptor molecules, such as the pesticide GLYP and Fusarium mycotoxins FB1 and BEA, could impact UHRF1 expression in bovine GC. Fusarium mycotoxins are natural contaminants of various commodities, such as cereal grain and animal feeds, and exposure to these mycotoxins has been linked to reproductive disorders in different species representing significant health problems worldwide (Schollenberger et al., 2007; Cortinovis et al., 2013; Albonico et al., 2016, 2017; El Khoury et al., 2019). BEA is a mycotoxin synthesized by several Fusarium spp. (Leslie and Summerell, 2006; Albonico et al., 2017) and acts as a severe cytotoxic agent against several mammalian cell lines (Klarić et al., 2008; Prosperini et al., 2012; Mallebrera et al., 2016). BEA’s main cytotoxic effects are mediated via its ability to promote transport of cations, such as calcium, through cell membranes disturbing normal physiological concentrations and thus affecting ionic homeostasis (Jestoi, 2008; Wang and Xu, 2012; Wu et al., 2018). Specifically, BEA was found to reversibly inhibit L-type calcium channels in a dose-dependent manner in a neuronal cell line (Wu et al., 2002). Interestingly, FSH-induced increases in GC calcium uptake are mediated via inhibition of L-type calcium channel activity (Peters et al., 2004; Kobayashi et al., 2006), but whether blockade of calcium channels is involved in BEA-induced inhibition of GC UHRF1 mRNA abundance will require further study. Previously, Albonico et al. (2017) reported that BEA decreases cell proliferation and steroid production in bovine GC and has dramatic inhibitory effects (i.e., >80% inhibition) on FSH plus IGF1-induced CYP11A1 and CYP19A1 mRNA abundance. In the present study, BEA completely inhibited FSH plus IGF1-induced UHRF1 expression, suggesting that this mycotoxin could also negatively affect reproductive success through the impairment of an epigenetic regulator such as UHRF1. No significant effect was observed on FSH plus IGF1-induced UHRF1 expression after exposure to GLYP and FB1 in the present study. Similarly, previous studies have shown that both GLYP (Perego et al., 2017) and FB1 (Albonico et al., 2017) had little or no effect on bovine GC proliferation or steroidogenesis. Collectively, the present and previous studies suggest that BEA is a potent environmental toxin that needs further study. Additional research is needed to better clarify: 1) E2F and cAMP regulation of UHRF1 gene expression within the ovary, 2) if UHRF1 is directly involved in the regulation of steroidogenesis and proliferation of follicular cells, and 3) the functional and epigenetic relevance and mechanism of action of UHRF1 in bovine GC and TC. It is important to emphasize that attempts to use Western blotting to detect UHRF1 protein in TC lysates in the present study were unsuccessful, most likely because of the small quantities of protein produced. Thus, additional developmental work will be required to ascertain if changes in cellular UHRF1 protein levels parallel UHRF1 mRNA levels during follicular growth and atresia. Conclusions UHRF1 expression in both GC and TC changes during follicle development, and its expression is regulated by environmental factors and several hormones and growth factors, including FGF9, steroids, and E2F transcription factors. An inverse relationship was observed between UHRF1 expression and global cellular DNA methylation levels in GC. How and if these changes in UHRF1 expression may alter the epigenome in OCs, alter the transgenerational epigenetic inheritance phenomenon, or are involved in the development of cystic ovaries will require further study. Abbreviations Abbreviations A4 androstenedione BEA beauvericin cAMP cyclic adenosine monophosphate Ct threshold cycle dbcAMP dibutyryl cAMP DEPC diethyl pyrocarbonate DHT dihydrotestosterone DMEM Dulbecco modified Eagle medium Dnase deoxyribonuclease DNMT1 DNA methyltransferase 1 E2 estradiol E2Fi E2F inhibitor EGF epidermal growth factor F12 Ham’s F-12 FB1 fumonisin B1 FCS fetal calf serum FGF9 fibroblast growth factor 9 FGFR FGF receptors FSH follicle-stimulating hormone Fsk forskolin GC granulosa cell GLYP glyphosate IGF1 insulin-like growth factor 1 LH luteinizing hormone OC oocyte P4 progesterone PCOS polycystic ovarian syndrome RING really interesting new gene siRNA small-interfering RNA TC theca cell UHRF1 ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 Acknowledgments We thank A. Hemple and J. Nichols for technical assistance; Dr. A. F. Parlow, National Hormone & Pituitary Program (Torrance, CA), for purified FSH and LH; and Creekstone Farms (Arkansas City, KS) for their generous donation of bovine ovaries. This research was supported in part by The Endowment of Howard M. & Adene R. Harrington Chair in Animal Science (Project 21-58500; to L.J.S.) and the Oklahoma Agricultural Experiment Station Project OKL02970 (approved for publication by the Director, Oklahoma Agricultural Experiment Station). Conflict of interest statement The authors declare no real or perceived conflicts of interest. Literature Cited Aad , P. Y. , S. E. Echternkamp, D. D. Sypherd, N. B. Schreiber, and L. J. Spicer. 2012 . The Hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: evidence of a link between the IGF and Hedgehog systems . Biol. Reprod . 87 : 79 . doi:10.1095/biolreprod.111.096735 Google Scholar Crossref Search ADS PubMed WorldCat Abbady , A. Q. , C. Bronner, K. Bathami, C. D. Muller, M. 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TI - Developmental and hormonal regulation of ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 gene expression in ovarian granulosa and theca cells of cattle
JO - Journal of Animal Science
DO - 10.1093/jas/skaa205
DA - 2020-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/developmental-and-hormonal-regulation-of-ubiquitin-like-with-plant-42rUnUu9iV
VL - 98
IS - 7
DP - DeepDyve
ER -