Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.

Isorhamnetin Protects Zearalenone-induced Damage via the PI3K/Akt Signaling Pathway in Porcine Granulosa Cells

Background: Zearalenone (ZEA) is widely derived from moldy cereal grain, which has adverse effects on animal reproduction. In particular, pigs are more sensitive to ZEA-induced toxicity than other animals. Isorhamnetin, a flavonoid has extensive of pharmacological activities. However, little is known about the effects of isorhamnetin on reproduction. Thus, it would be interesting to clarify the effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced cytotoxicity in porcine granulosa cells.Results: Our findings showed that isorhamnetin suppressed ZEA-induced apoptosis by regulating Bcl-2 and Bax protein changes. Changes in intracellular Ca2+ levels and CHOP, ATF6, GRP78 indicated that isorhamnetin rescued ZEA-induced endoplasmic reticulum stress (ERS). Furthermore, isorhamnetin prevented ZEA-induced reactive oxygen species (ROS) via P38 signaling pathway. Mechanistically, isorhamnetin stimulated the expression of PCNA and CyclinD, thereby raising the ratio of S phases cells in response to ZEA-induced apoptosis via PI3K/Akt signaling pathway. Isorhamnetin also recovered ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins (FSH-R, CYP19A1). Conclusions: Collectively, these findings show that isorhamnetin protects granulosa cells from ZEA-induced damage via the PI3K/Akt signaling pathway, which promotes proliferation, alleviates steroidogenesis disorder, ERS and oxidative stress.

MicroRNA-21 enhances estradiol production by inhibiting WT1 expression in granulosa cells

In antral follicles, transition of proliferative granulosa cells to estradiol-producing is critical for proper oocyte maturation. MicroRNAs are noncoding RNAs that play important roles in ovarian follicular development, however this has yet to be fully characterized. MicroRNA-21 is significantly higher in granulosa cells isolated from large antral follicles compared to those from small antral follicles. To investigate the function of miR-21, porcine granulosa cells were transfected with miR-21 mimic or miR-21 targeted siRNA. Cells with the miR-21 mimic had higher aromatase expression and estradiol production but decreased WT1 expression. Conversely, cells with the miR-21 siRNA secreted less estradiol and had higher WT1 expression. We hypothesized miR-21 promotes estradiol production by inhibiting WT1 protein synthesis. We found a potential miR-21 binding site in the 3’UTR of the WT1 transcript and performed a dual luciferase reporter assay using the wild-type and mutated 3’UTR. Compared to the negative control, the miR-21 mimic induced a significant decrease in luciferase activity in the wild-type 3’UTR. This decrease was reversed when the 3’UTR was mutated, suggesting miR-21 targets this site to inhibit WT1 expression. We next transfected porcine granulosa cells with WT1 targeted siRNA and observed a significant increase in aromatase expression and estradiol secretion. We propose that miR-21 represses WT1 expression in granulosa cells to potentially promote aromatase expression and estradiol production. This study offers the first report of a microRNA regulating WT1 expression in granulosa cells and reveals the role of miR-21 in WT1’s regulation of estradiol production.

FGF21 Promotes Proliferation and Estradiol Synthesis in Porcine Granulosa Cells

Background: The proliferation and estradiol synthesis in granulosa cells (GCs) directly promotes follicular development. Previous studies had found that FGF21 regulated the hypothalamic-pituitary-gonad axis in response to the control of fertility. However, the functions and mechanisms of FGF21 in GCs are unclear.Results: Here, we found that the mRNA and protein levels of FGF21 in the ovarian tissue of high-yielding sows (Sus scrofa) was higher than that in low-yielding sows. Moreover, FGF21 was predominantly expressed in porcine GCs. Additionally, ELISA assay showed estradiol was significantly increased when overexpression of FGF21 in porcine GCs. Meanwhile, overexpressed FGF21 up-regulated both the mRNA and protein levels of key estradiol synthesis-related genes in porcine GCs, including StAR, CYP11A1 and CYP19A1. Corresponsingly, knockdown FGF21 inhibited estradiol levels and its synthesis-related genes expression. Besides, overexpression of FGF21 promoted the proliferation of porcine GCs, displayed as increasing the percentage of S-phase cells in cell cycle and EdU positive cells, including cell viability, and upregulated cell cycle genes, including cell cycle protein B (Cyclin B) and protein E (Cyclin E). Corresponsingly, knockdown FGF21 in porcine GCs suppressed the cell cycle and cell viability, as well as EdU positive cell number.Conclusions: These findings highlight that FGF21 is associated with the development of GCs and may be a novel underlying regulator of porcine follicular development.

The Effect of RBP4 on microRNA Expression Profiles in Porcine Granulosa Cells

Retinol binding protein 4 (RBP4) is a transporter of vitamin A that is secreted mainly by hepatocytes and adipocytes. It affects diverse pathophysiological processes, such as obesity, insulin resistance, and cardiovascular diseases. MicroRNAs (miRNAs) have been reported to play indispensable roles in regulating various developmental processes via the post-transcriptional repression of target genes in mammals. However, the functional link between RBP4 and changes in miRNA expression in porcine granulosa cells (GCs) remains to be investigated. To examine how increased expression of RBP4 affects miRNA expression, porcine GCs were infected with RBP4-targeted lentivirus for 72 h, and whole-genome miRNA profiling (miRNA sequencing) was performed. The sequencing data were validated using real-time quantitative polymerase chain reaction (RT-qPCR) analysis. As a result, we obtained 2783 known and 776 novel miRNAs. In the experimental group, 10 and seven miRNAs were significantly downregulated and upregulated, respectively, compared with the control group. Ontology analysis of the biological processes of these miRNAs indicated their involvement in a variety of biological functions. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that these miRNAs were involved mainly in the chemokine signaling pathway, peroxisome proliferators-activated receptors (PPAR) signaling pathway, insulin resistance pathway, nuclear factor-kappa B(NF-kappa B) signaling pathway, and steroid hormone biosynthesis. Our results indicate that RBP4 can regulate the expression of miRNAs in porcine GCs, with consequent physiological effects. In summary, this study profiling miRNA expression in RBP4-overexpressing porcine GCs provides an important reference point for future studies on the regulatory roles of miRNAs in the porcine reproductive system.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = −6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.