Bovine Ovarian Granulosa Cells Research Articles

Função do fator de crescimento fibroblástico 18 na regulação da cascata de eventos pré-ovulatórios em bovinos

ABSTRACT: Suboptimal LH surges can lead to delayed or absent ovulation during reproductive management programs in cattle. Several signaling pathways may enhance the LH signal, potentially including growth factors. In the present study, we determined whether Fibroblast growth factor 18 (FGF18), which mimics some actions of the LH surge on granulosa cells, may potentiate the action of a suboptimal dose of LH using an established bovine granulosa cell (GC) culture system. Addition of recombinant FGF18 increased the concentration of progesterone (P4) in the culture medium. A combination of LH and FGF18 in the culture medium increased the abundance of mRNA encoding EREG, EGR1 and EGR3, proteins that are early modulators of the LH-induced pre-ovulatory cascade. This study demonstrated that FGF18 may be a co-factor in the regulation of the pre-ovulatory cascade in bovine ovarian granulosa cells.

Melatonin Attenuates Oxidative Stress-Induced Apoptosis of Bovine Ovarian Granulosa Cells by Promoting Mitophagy via SIRT1/FoxO1 Signaling Pathway.

Oxidative-stress-induced apoptosis of granulosa cells is considered to be a main driver of follicular atresia. Increasing evidence suggests a protective effect of melatonin against oxidative damage but the mechanism remains unclear. The aim of this study is to investigate the effects of melatonin on mitophagy and apoptosis of bovine ovarian granulosa cells under oxidative stress, and to clarify the mechanism. Our results indicate that melatonin inhibited H2O2-induced apoptosis and mitochondrial injury of bovine ovarian granulosa cells, as revealed by decreased apoptosis rate, reactive oxygen species (ROS) levels, Ca2+ concentration, and cytochrome C release and increased mitochondrial membrane potential (ΔΨm). Simultaneously, melatonin promoted mitophagy of bovine ovarian granulosa cells through increasing the expression of PTEN-induced putative kinase 1 (PINK1), PARKIN, BECLIN1, and LC3II/LC3I; decreasing the expression of sequestosome 1 (SQSMT1); and promoting mitophagosome and lysosome fusion. After treatment with a mitophagy inhibitor CsA, we found that melatonin alleviated apoptosis and mitochondrial injury through promoting mitophagy in bovine ovarian granulosa cells. Furthermore, melatonin promoted the expression of silent information regulator 1 (SIRT1) and decreased the expression level of forkhead transcription factors class O (type1) (FoxO1). By treatment with an SIRT1 inhibitor EX527 or FoxO1 overexpression, the promotion of melatonin on mitophagy as well as the inhibition on mitochondrial injury and apoptosis were reversed in bovine ovarian granulosa cells. In conclusion, our results suggest that melatonin could promote mitophagy to attenuate oxidative-stress-induced apoptosis and mitochondrial injury of bovine ovarian granulosa cells via the SIRT1/FoxO1 signaling pathway.

Chia (Salvia hispanica L.) can directly suppress basic ovarian cell functions in two farm animal species and protect ovarian cells from the proliferation-stimulating influence of xylene.

The influence of the functional food plant chia (Salvia hispanica L.) on reproduction functions and its ability to prevent the negative effects of environmental contaminants has not yet been studied. Our study aimed to examine the effect of chia seed extract alone and in combination with xylene on the markers of proliferation, apoptosis and hormones release by cultured bovine and porcine ovarian granulosa cells. The extract of chia reduced all of the measured parameters in bovine and porcine ovarian cells but had no effect on the proliferation of porcine cells. Xylene, stimulated proliferation and IGF-I release and inhibited the release of progesterone and testosterone but not apoptosis of bovine granulosa cells. It promoted proliferation, apoptosis and progesterone output by porcine cells. Chia mitigated the stimulatory effect of xylene on proliferation but not on other parameters in both species. The present results are the first demonstration of a direct effect of chia on basic ovarian cell functions. They confirmed a direct influence of xylene on these functions and found a similar stimulatory action of xylene on bovine and porcine ovarian cell proliferation. The present observations demonstrated species-specific differences in the characteristics of xylene influences on ovarian cell apoptosis and secretory activity. Finally, the present results indicate that chia can be a natural protector against the proliferation-stimulating effects of xylene on ovarian cells in both species.

Protective effects of MNQ against Lipopolysaccharide-induced inflammatory damage in bovine ovarian follicular granulosa cells in Vitro

Inflammation of the reproductive tract in dairy cows lead to functional disorders of follicular granulosa cells (GCs) in mammalian ovaries resulting in infertility and serious losses to the livestock industry. Lipopolysaccharide (LPS) can induce an inflammatory response in follicular granulosa cells in vitro. The aim of this study was to investigate the cellular regulatory mechanism of MNQ (2-methoxy-1,4-naphthoquinone) on eliminating the inflammatory response and restoring normal functions for bovine ovarian follicular GCs cultured in vitro exposed to LPS. The cytotoxicity of MNQ and LPS on GCs were detected by MTT method to determine the safe concentration. The relative expression of inflammatory factors and steroid synthesis-related genes were detected by qRT-PCR. The concentration of steroid hormones in the culture broth were detected by ELISA. Differential gene expressions were analyzed by RNA-seq. There were no toxic effects on GCs at MNQ and LPS concentrations of less than 3 µM and 10 µg/mL, respectively and treated in 12 h. The relative expressions of IL-6, IL-1β and TNF-α were significantly higher in the LPS group compared with the CK group when GCs cultured in vitro were treated with the above concentrations and times (P < 0.05), but significantly lower in the MNQ+LPS group compared with the LPS group (P < 0.05). The levels of E2 and P4 in the culture solution were significantly reduced in the LPS group compared to the CK group (P < 0.05), and restored in the MNQ+LPS group. The relative expressions of CYP19A1, CYP11A1, 3β-HSD, and STAR were significantly decreased in the LPS group compared with the CK group (P < 0.05), while the MNQ+LPS group also recovered to some extent. There were 407 differential genes shared by LPS vs CK and MNQ+LPS vs LPS by RNA-seq analysis, which were mainly enriched in steroid biosynthesis and TNF signaling pathway. We screened 10 genes for analysis and found consistent results for RNA-seq and qRT-PCR. In this study, we confirmed the protective effect of MNQ, an extract from Impatiens balsamina L, on LPS-induced inflammatory responses in bovine follicular granulosa cells in vitro as well as functional damage, and acted through steroid biosynthesis and TNF signaling pathways.

High mobility group box-1 regulates expression of EGFR, VEGF, StAR and TIMP1/2 in bovine granulosa cells through a mechanism involving TLR2/NF-κB

Innate immunity is involved in ovarian activity through aseptic inflammation and tissue repair. High-mobility group box-1 (HMGB1) is related to placental inflammation and a driver of inflammation throughout pregnancy. The aim of this study was to investigate the interaction of HMGB1 with TLR2 in bovine ovarian granulosa cells, and the effects of HMGB1 on bovine ovarian granulosa cells in vitro. The viability of granulosa cells were not affected by HMGB1 with the concentration less than 5 μg/mL. The mRNA levels of TLR2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), the steroidogenic acute regulatory protein (StAR), tissue inhibitors of matrix (TIMP1/2) of ovarian granulosa cells were upregulated by HMGB1(P < 0.05). The protein levels of TLR2, TLR1 and phosphorylation-NF-κB (p-NF-κB) p65 in ovarian granulosa cells increased in 5 μg/mL HMGB1 group (P < 0.05), and TLR2 decreased in siRNA-2 group (P < 0.05). IL-6 of ovarian granulosa cells was increased by 1 μg/mL and 5 μg/mL HMGB1 (P < 0.05). These results implicate that HMGB1 has interaction with TLR2, TLR1 and p-NF-κB p65 in ovarian granulosa cells, which lead to nuclear translocation of NF-κB p65 and the secretion of interleukin-6 (IL-6). HMGB1 regulates the expression of EGFR, VEGF, StAR, TIMP1/2 and the secretion of IL-6 in ovarian granulosa cells of dairy cows through activating the TLR2/NF-κB signaling pathway, which may be interfere with ovarian physiological activity.

Dimethyl itaconic acid improves viability and steroidogenesis and suppresses cytokine production in LPS-treated bovine ovarian granulosa cells by regulating TLR4/nfkβ, NLRP3, JNK signaling pathways

The stimulation of pro-inflammatory pathways by lipopolysaccharide (LPS) endotoxins is a key player in the pathological mechanisms involved in the development of ovarian dysfunctions in dairy cows. Dimethyl itaconate acid (DMIA) is a novel immunometabolite that has recently emerged as a regulator of inflammatory responses in mammals. The present study was undertaken to determine the anti-inflammatory effects of DMIA on bovine granulosa cells (GCs) and to explore its possible molecular mechanisms.The ovarian GCs were obtained from small follicles of dairy cows. The GCs were stimulated with 1 μg/mL LPS for 4 h and then treated with 250 μM DMIA for 12 h. The viability, production of pro-inflammatory cytokines, activation of inflammatory signaling pathways and synthesis of steroid hormones were evaluated in treated GCs.Our results showed that DMIA reduced the inflammatory responses in LPS stimulated GCs by down-regulating the expression of nod-like receptor family pyrin domain containing 3 inflammasome, and toll-like receptor 4 and by suppressing the phosphorylation of nuclear factor kappa B and c-Jun N-terminal kinase proteins. DMIA also attenuated the increased production of pro inflammatory cytokines (interleukin 6, tumor necrosis factor α and interleukin 1 beta (p < 0.01) in LPS stimulated GCs. Exposure of LPS stimulated GCs to DMIA improved the impaired steroidogenesis by up-regulation of steroid synthesis genes including 3-beta-hydroxysteroid dehydrogenase, follicle stimulating hormone receptor and cytochrome P450 aromatase.The results of the present study highlight the potential role of itaconic acid for the improvement of GCs inflammation in dairy cows with ovarian dysfunctions.

Tribulus terrestris can suppress the adverse effect of toluene on bovine and equine ovarian granulosa cells.

Influence of oil-related product toluene and herbal remedy puncturevine Tribulus terrestris L. (TT) on female reproduction is known. Yet, mechanisms of their action on ovaries in different species and potential protective effect of TT against adverse toluene action remain to be established. We studied the effect of toluene, TT, and their combination on ovarian granulosa cells from two mammalian species (cows and horses). Viability, markers of proliferation (PCNA) and apoptosis (bax), steroid hormones, IGF-I, oxytocin, and prostaglandin F (PGF) release were analyzed by trypan blue exclusion test, quantitative immunocytochemistry, and EIA/ELISA. Toluene suppressed all analyzed parameters. In both species, TT stimulated proliferation and reduced progesterone, oxytocin, and PGF. In horses, TT inhibited testosterone and IGF-I. In both species, TT supported toluene effect on viability, steroids, IGF-I, and PGF, and inverted its action on apoptosis. In cows, TT promoted toluene effect on proliferation. In horses, TT supported toluene effect on oxytocin but suppressed its influence on proliferation. In both species, toluene induced inhibitory action of TT on viability, steroids, IGF-I, and PGF, and prevented its stimulatory action on proliferation. In cows, toluene supported inhibitory action of TT on oxytocin and prevented its stimulatory action on apoptosis. In horses, toluene induced stimulatory effect of TT on apoptosis. Our results indicate potential toxic toluene effect on farm animal ovaries, applicability of TT as a biostimulator of farm animal reproduction and as a protector against the adverse influence of toluene on female reproduction.

Expression of ERβ induces bovine ovarian granulosa cell autophagy via the AKT/mTOR pathway.

The aim of our study was to determine whether silencing or overexpression of estrogen receptor β (ERβ) regulates cell proliferation, steroidogenesis, autophagy and signalling pathways in bovine ovarian granulosa cells in vitro. In this study, bovine ovarian granulosa cells (BGCs) were cultured and transfected with ERβ siRNA (si-ERβ) or a plasmid overexpressing ERβ (oe-ERβ), and CCK-8 kit was used to assess cell proliferation. Real-time PCR was used to measure gene transcription. Western blotting was used to measure protein expression, and a specific kit was used to measure the production of steroid hormones. The results showed the expression level of ERβ affects BGC proliferation according to the gene transcription levels of FSHR, CYP19A1, HSD3β1 and STAR and the production of E2 and P4. ERβ was identified as an important nuclear receptor that induced BGC autophagy based on the mRNA and protein expression of autophagy-related genes. Furthermore, the role of ERβ in BGC autophagy was confirmed through treatment with rapamycin (RAPA) or 3-methyladenine (3-MA) in BGCs by cotransfection with si-ERβ or oe-ERβ in BGCs. The results related to AKT/mTOR signalling and phosphorylation suggested that ERβ induces BGC autophagy through attenuating AKT/mTOR signalling. In summary, this study demonstrates that silencing or overexpression of ERβ regulates BGC proliferation and function and induces BGC autophagy by targeting AKT/mTOR signalling. These data reveal a novel regulatory mechanism of autophagy via ERβ and provide insights into the role of autophagy in BGCs.

Liver Receptor homolog-1 Regulates Apoptosis of Bovine Ovarian Granulosa Cells by Progestogen Receptor Signaling Pathway.

Simple SummaryLiver receptor homolog-1 (LRH-1) is highly observed in tissues with secretory function, such as the ovaries, suggesting that LRH-1 may play an essential role in the ovarian endocrine. In particular, ovarian granulosa cells (GCs) are the functional cells that produce steroid hormones. The fate of GCs directly affects follicular development or atresia. These effects were lost in granulosa-specific LRH-1-knockout mice, showing that LRH-1 is a central modulator of ovarian function. However, the underlying mechanism LRH-1 in the bovine ovaries remains unclear. We aimed to detect the effect of LRH-1 on steroid hormones in bovine GCs and explore the potential relationship between LRH-1 and the fate of GCs. The results show that LRH-1 was specifically highly expressed in GCs of atretic follicles. Mechanistically, LRH-1 induced the apoptosis of bovine GCs by the progestogen receptor signaling pathway. While this finding provided new ideas for the study of follicular atresia, it also provided a theoretical basis for the clinical diagnosis and treatment for infertility in cow.The purpose of the present investigation was to assess the function of LRH-1 on GCs and the mechanisms involved. Here, LRH- was highly expressed in the bovine GCs of atretic follicles. Treatment with 50 μM of LRH-1 agonist (DLPC) significantly induced the expression of LRH-1 (p < 0.05). In particular, LRH-1 activation blocked the progestogen receptor signaling pathway via downregulating progesterone production and progestogen receptor levels (p < 0.05), but had no effect on 17 beta-estradiol synthesis. Meanwhile, LRH-1 activation promoted the apoptosis of GCs and increased the activity of caspase 3 (p < 0.05). Importantly, upregulating the progestogen receptor signaling pathway with progestogen could attenuate the LRH-1-induced proapoptotic effect. Moreover, treatment with progestogen decreased the activity of the proapoptotic gene caspase 3 and increased the expression of antiapoptotic gene Bcl2 in LRH-1 activated GCs (p < 0.05). Taken together, these results demonstrate that LRH-1 might be dependent on the progestogen receptor signaling pathway to modulate bovine follicular atresia.

Potential protective effect of puncturevine (Tribulus terrestris, L.) against xylene toxicity on bovine ovarian cell functions.

The action of the medicinal plant Tribulus terrestris (TT) on bovine ovarian cell functions, as well as the protective potential of TT against xylene (X) action, remain unknown. The aim of the present in vitro study was to elucidate the influence of TT, X and their combination on basic bovine ovarian cell functions. For this purpose, we examined the effect of TT (at doses of 0, 1, 10, and 100 ng/mL), X (at 20 ?g/mL) and the combination of TT + X (at these doses) on proliferation, apoptosis and hormone release by cultured bovine ovarian granulosa cells. Markers of proliferation (accumulation of PCNA), apoptosis (accumulation of Bax) and the release of hormones (progesterone, testosterone and insulin-like growth factor I, IGF-I) were analyzed by quantitative immunocytochemistry and RIA, respectively. TT addition was able to stimulate proliferation and testosterone release and inhibit apoptosis and progesterone output. The addition of X alone stimulated proliferation, apoptosis and IGF-I release and inhibited progesterone and testosterone release by ovarian cells. TT was able to modify X effects: it prevented the antiproliferative effect of X, induced the proapoptotic action of X, and promoted X action on progesterone but not testosterone or IGF-I release. Taken together, our observations represent the first demonstration that TT can be a promoter of ovarian cell functions (a stimulator of proliferation and a suppressor of apoptosis) and a regulator of ovarian steroidogenesis. X can increase ovarian cell proliferation and IGF-I release and inhibit ovarian steroidogenesis. These effects could explain its anti-reproductive and cancer actions. The ability of TT to modify X action on proliferation and apoptosis indicates that TT might be a natural protector against some ovarian cell disorders associated with X action on proliferation and apoptosis, but it can also promote its adverse effects on progesterone release.

26 The Effect of Cannabidiol on Follicular Growth and Ovulation in Cattle

Abstract The role of the endocannabinoids in regulating female reproductive processes such as follicular development has become an area of heightened interest with the prevalence of Cannabidiol (CBD) increasing as a natural mediator to physiological issues as well as the addition of hemp meal as a feedstuff for livestock. Initial studies indicate that cannabis use including Δ 9- tetrahydrocannabinol (THC) has negative implications on folliculogenesis and multiple aspects of pregnancy. However, there is limited knowledge on the effect of CBD on follicular growth and ovulation. The objective of this study was to identify the presence of the endocannabinoid system (ECS) in bovine ovarian theca and granulosa cells and its effect on follicular dynamics leading to ovulation. Theca and granulosa cells from medium (&amp;lt; 4 mm) and large ( &amp;gt;4 mm) follicles from abattoir bovine ovaries (n=61) were collected. Upon completion of pooling theca and granulosa cells, protein was isolated for detection of cannabinoid receptors, type 1 (CB1) and type 2 (CB2) through western blot. Receptors CB1 and CB2 are G- protein coupled receptors with an effect on the adenylyl cyclase pathway, which is key to many reproductive functions in the ovary. A strong visual representation of CB1 receptor detection was found in large granulosa cells and theca cells. Using Stain-Free total protein measurement as the loading control, the densitometric ratio in medium granulosa cells and large granulosa cells had a greater abundance of CB2 (P &amp;lt; 0.05) compared to theca cells. This data suggests a detectable effect by cannabinoids on granulosa and theca cells. Future studies are needed to investigate the role the ECS has in folliculogenesis and key steroidal production for successful ovulation. The subsequent knowledge is a key first step in understanding the effect CBD has on fertility and pregnancy in livestock.

Activin A attenuates apoptosis of granulosa cells in atretic follicles through ERβ‐induced autophagy

It is well known that approximately 99% of ovarian follicles in mammals suffer from a degenerative process known as atresia, which is a huge waste of genetic resource in female animals. Studies have shown that activin A (ACT-A) is located in ovarian granulosa cells and has different effects in granulosa cell depending on species. Although granulosa cells play a critical role during follicular atresia, the mechanism of action of ACT-A in bovine ovarian granulosa cells (BGC) is poorly understood. In this study, we first determined the apoptosis of BGCs isolated from growth follicles and atretic follicles respectively. Then, BGC isolated from atretic follicles were used as a model to elucidate the role of ACT-A in cattle ovary. The results showed that apoptosis occurred in both growing follicles and atretic follicles, and the percentage of apoptotic cells in atretic follicles was higher than that in growing follicles. The current results indicated that ACT-A can attenuate apoptosis of BGC by maintaining the function of BGC in atretic follicles. Increased ERβ induced by ACT-A promoted BGC autophagy but had no effect on apoptosis. In summary, this study suggests that ACT-A attenuates BGC apoptosis in atretic follicles by ERβ-mediated autophagy signalling.

MicroRNA-10b promotes the apoptosis of bovine ovarian granulosa cells by targeting plasminogen activator inhibitor-1

Granulosa cells (GCs) are essential somatic cells in the ovaries, and apoptosis of GCs causes follicular atresia. microRNA-10b (miR-10b) is pivotal for cell apoptosis. However, currently, little is known about the role of miR-10b in bovine ovarian GCs (BGCs). In this study, the effect of miR-10b on the apoptosis of BGCs was investigated. Our results showed that the overexpression of miR-10b could increase the apoptosis rate of BGCs, which is associated with the increased expression of Caspase-3 and decreased expression ratio of Bcl-2/Bax (P < 0.05). Furthermore, plasminogen activator inhibitor-1 (PAI-1) was confirmed to be a validated target of miR-10b in BGCs using dual-luciferase reporter analysis, and transfection of miR-10b mimics decreased the expression of PAI-1 (P < 0.05). In addition, overexpression of PAI-1 significantly inhibited BGC apoptosis (P < 0.05), and PAI-1 could alleviate BGC apoptosis induced by miR-10b (P < 0.05). Subsequently, phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) was found to be the downstream pathway of PAI-1 by RNA-Seq analysis and verified by Western blot. Finally, a PI3K/AKT inhibitor (Miltefosine) was used to inhibit the PI3K/AKT pathway, which reversed the inhibitory effect of PAI-1 on the apoptosis of BGCs (P < 0.05), and enhanced the promotion effect of miR-10b on the apoptosis of BGCs (P < 0.05). Our results indicated that miR-10b promotes BGC apoptosis by targeting PAI-1 to regulate the PI3K/AKT pathway.

Vitamin E and selenium supplementation synergistically alleviate the injury induced by hydrogen peroxide in bovine granulosa cells

Dairy cows are susceptible to reproductive disorders, which are thought to be associated with oxidative stress. In the study, we investigated the effects of vitamin E (VE) and selenium (Se) on the proliferation, apoptosis, and steroidogenesis in bovine ovarian granulosa cells under hydrogen peroxide (H2O2) – induced oxidative stress and elaborated the underlying mechanisms. Our results showed that VE or Se could stimulate the granulosa cell proliferation, possibly due to up-regulating the expression of CCND1 and decreasing the P21 levels under oxidative stress. VE or Se treatment also increased the secretion of estradiol (E2) and progesterone (P4), which could be owing to improving the expression of genes associated with steroidogenesis (StAR, HSD3β1, and CYP19A1) expression. VE or Se treatment down-regulated the apoptosis-related genes (BAX, CASP3) expression and decreased cell apoptosis. Furthermore, VE or Se treatment inhibited reactive oxidative species (ROS) and malondialdehyde (MDA) generation, increased total antioxidant capacity (T-AOC), and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Additionally, VE or Se treatment also alleviated the endoplasmic reticulum stress, activated the nuclear factor erythroid 2-related factor 2 (NRF2), and up-regulated the expression of its downstream genes, including NQO1, HO-1, GCLM, GCLC. More importantly, compared with either VE or Se treatment alone, their combined treatment showed a better protective effect against oxidative damage. Overall, our results indicated that VE and Se synergistically stimulated the granulosa cell proliferation and steroidogenesis, decreased cell apoptosis, mitigated the endoplasmic reticulum stress by activating the NRF2 signal pathway.

High doses of FSH induce autophagy in bovine ovarian granulosa cells via the AKT/mTOR pathway.

Follicle-stimulating hormone (FSH) plays a critical role in follicular growth and granulosa cell function; however, the mechanism by which the aggressive stimulation of FSH leads to poorer oocyte quality and embryo development potential is unclear. In this study, bovine ovarian granulosa cells (BGCs) were challenged with FSH doses (vehicle, 0.1, 1, 10 and 100ng/ml) to investigate the effects of FSH on BGCs. The results indicated that the relative viability of BGCs was significantly increased in cells challenged with 1ng/ml FSH, whereas the viability was significantly decreased with 100ng/ml FSH treatment. The mRNA abundance of FSHR, CYP19, StAR and BAX was significantly upregulated with 1, 10 and 100ng/ml of FSH, while the BCL-2 mRNA level was downregulated with higher concentrations of FSH (10 and 100ng/ml). Furthermore, BGC autophagy was detected in cells treated with 10 and 100ng/ml FSH by MDC staining, and the mRNA abundance of LC3, BECN1, BNIP3, ATG3 and ATG7 was upregulated with increasing FSH concentration. Meanwhile, the protein expression of LC3 was increased in cells treated with 10 and 100ng/ml FSH. 1 and 10ng/ml FSH significantly increased E2 production, whereas 10 and 100ng/ml FSH significantly increased P4 production. FSH significantly inhibited the phosphorylation of AKT in cells treated with higher concentrations (1, 10 and 100ng/ml), while activating mTOR phosphorylation at concentrations of 10 and 100ng/ml of FSH. In summary, we can conclude that higher doses of FSH (10 and 100ng/ml) induce BGC autophagy via the AKT/mTOR signalling pathway.

WITHDRAWN:Estrogen receptor β (ERβ) induces bovine ovarian granulosa cell (BGC) autophagy via the AKT/mTOR pathway.

Ahead of Print article withdrawn by Editorial Board.

MiRNA-21–3p targeting of FGF2 suppresses autophagy of bovine ovarian granulosa cells through AKT/mTOR pathway

It is widely thought that the main reason for ovarian follicular atresia is apoptosis of granulosa cells, however, accumulating evidence suggests that autophagy plays a role in the fate of granulosa cells. Although epigenetic regulation including miR-21–3p associated with autophagy process has been reported in many cancer types, nevertheless, the mechanism of miR-21–3p in bovine ovary is poorly understood. In the present study, bovine ovarian granulosa cells (BGCs) were used as a model to elucidate the autophagy and role of miR-21–3p in a cattle ovary. The results from gene expression and tagged autophagosomes showed the autophagy in BGCs and miR-21–3p was identified as an important miRNA regulating autophagy of BGCs. The current results indicated that FGF2 was a validated target of miR-21–3p in autophagy regulation of BGCs according to the results from FGF2 luciferase reporter assays and FGF2 overexpression (oe-FGF2) or small interference (si-FGF2). Transfection of miR-21–3p mimic and si-FGF2 plasmids resulted in decreasing phosphorylated AKT and mTOR, while transfection of miR-21–3p inhibitor and oe-FGF2 increased the phosphorylated level of AKT and mTOR in BGCs. These data indicate that regulation of miR-21–3p on BGCs autophagy through AKT/mTOR pathway. In summary, this study suggests that miR-21–3p targets FGF2 to inhibit BGCs autophagy by repressing AKT/mTOR signaling.

Functional effects of Tribbles homolog 2 in bovine ovarian granulosa cells†.

Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6h through 24h post-hCG as compared to 0h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.

UFL1 Alleviates LPS-Induced Apoptosis by Regulating the NF-κB Signaling Pathway in Bovine Ovarian Granulosa Cells.

Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) is an E3 ligase of ubiquitin fold modifier 1 (UFM1), which can act together with its target protein to inhibit the apoptosis of cells. Lipopolysaccharides (LPS) can affect the ovarian health of female animals by affecting the apoptosis of ovarian granulosa cells. The physiological function of UFL1 on the apoptosis of bovine (ovarian) granulosa cells (bGCs) remains unclear; therefore, we focused on the modulating effect of UFL1 on the regulation of LPS-induced apoptosis in ovarian granulosa cells. Our study found that UFL1 was expressed in both the nucleus and cytoplasm of bGCs. The results here demonstrated that LPS caused a significant increase in the apoptosis level of bGCs in cows, and also dramatically increased the expression of UFL1. Furthermore, we found that UFL1 depletion caused a significant increase in apoptosis (increased the expression of BAX/BCL-2 and the activity of caspase-3). Conversely, the overexpression of UFL1 relieved the LPS-induced apoptosis. In order to assess whether the inhibition of bGCs apoptosis involved in the nuclear factor-κB (NF-κB) signaling pathway resulted from UFL1, we detected the expression of NF-κB p-p65. LPS treatment resulted in a significant upregulation in the protein concentration of NF-κB p-p65, and knockdown of UFL1 further increased the phosphorylation of NF-κB p65, while UFL1 overexpression significantly inhibited the expression of NF-κB p-p65. Collectively, UFL1 could suppress LPS-induced apoptosis in cow ovarian granulosa cells, likely via the NF-κB pathway. These results identify a novel role of UFL1 in the modulation of bGC apoptosis, which may be a potential signaling target to improve the reproductive health of dairy cows.

MiR-21-3p inhibits autophagy of bovine granulosa cells by targeting VEGFA via PI3K/AKT signaling.

It is well documented that granulosa cell apoptosis is the main reason for follicular atresia and death; however, increasing evidence suggests that autophagy plays an important role in the fate of granulosa cells. miR-21-3p regulates many fundamental biological processes and is pivotal in the autophagy of tumor cells; nevertheless, the autophagy in cattle ovary and how miR-21-3p regulates the follicular cells is unknown. In this study, we aimed to elucidate the autophagy and the role of miR-21-3p in cattle ovary using bovine primary ovarian granulosa cells (BGCs). The results showed the autophagy for the first time in BGCs in large follicle according to autophagic gene transcript of LC3, BECN-1, ATG3, protein expression of LC3, P62 and LC3 puncta, a standard marker for autophagosomes. miR-21-3p was identified as a novel miRNA that repressed BGCs autophagy according to the results from plasmids transfection of miR-21-3p mimics and inhibitor. Meanwhile, VEGFA was confirmed to be a validated target of miR-21-3p in BGCs using luciferase reporter assays and the results of VEGFA expression decreased with transfection of miR-21-3p mimics, while it increased with transfection of miR-21-3p inhibitor. In addition, small interference-mediated knockdown of VEGFA significantly inhibits BGCs autophagy signaling; however, overexpression of VEGFA in BGCs promoted autophagy in the presence of miR-21-3p. Finally, the results of AKT and its phosphorylation suggested that miR-21-3p suppressed VEGFA expression through downregulating AKT phosphorylation signaling. In summary, this study demonstrates that miR-21-3p inhibits BGCs autophagy by targeting VEGFA and attenuating PI3K/AKT signaling.