Steroidogenesis In Granulosa Cells Research Articles

Effect of heat exposure on the growth and developmental competence of bovine oocytes derived from early antral follicles

In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte–cumulus–granulosa complexes (OCGCs) derived from early antral follicles (0.5–1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.

Bisphenol S Alters the Steroidome in the Preovulatory Follicle, Oviduct Fluid and Plasma in Ewes With Contrasted Metabolic Status.

Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.

Integrated Proteomic and Metabolomic Analyses of Chicken Ovary Revealed the Crucial Role of Lipoprotein Lipase on Lipid Metabolism and Steroidogenesis During Sexual Maturity.

During sexual maturation and ovulatory cycle in chickens, ovaries undergo dynamic morphological and functional changes. The aim of this study was to evaluate the integrated proteome and metabolome analyses of chicken ovaries to characterize the changes in protein and metabolite profiles during sexual maturity. The ovary of Rohman layers before (125 days of age) and after (139 days of age) sexual maturation were collected for proteome and metabolome sequencing. The results showed that a total of 680 differentially expressed proteins (DEPs) and 1,046 differential metabolites (DMs) were identified in the chicken ovary during sexual maturity. Among the DEPs, 595 proteins were up-regulated and 85 were down-regulated, whereas 519 metabolites were up-regulated and 527 were down-regulated. KEGG pathway enrichment analysis showed that DEPs were significantly enriched in glycerolipid metabolism, calcium signaling pathway, folate biosynthesis, fat digestion and absorption, NF-kB signaling pathway, and PPAR signaling pathway. However, DMs were significantly enriched in the metabolism pathways, PPAR signalling pathway, glycerolipid metabolism, ferroptosis, biosynthesis of amino acids, and biosynthesis of unsaturated fatty acids. The results of the integrated analyses of DEPs and DMs revealed that the PPAR signaling pathway and glycerolipid metabolism were the most significantly enriched pathways. Among the identified DEPs, lipoprotein lipase (LPL) was upregulated in sexually mature chicken ovaries and was significantly enriched in the glycerolipid metabolism pathway, which may partially explain the possible reasons for steroidogenesis and lipid reserves responsible for oocyte maturation and ovarian follicle development during sexual maturity in chickens. The results further revealed that LPL silencing decreased the content of lipid droplets (LDs), as well as the mRNA expression of lipid metabolism-related genes including; sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase (FASN); and steroidogenesis-related genes such as; cytochrome P450 11A1 (CYP11A1) and steroidogenic acute regulatory (StAR). The present study revealed that upregulation of LPL in the chicken ovary during sexual maturity promotes granulosa cell (GC) lipid metabolism and steroidogenesis. These findings provide a theoretical support for further studies to elucidate the mechanism of lipid metabolism to regulate the function of avian GCs during sexual maturity in chickens.

The Role of Nitric Oxide on Male and Female Reproduction.

Nitric oxide (NO), a reactive nitrogen species, is a molecule of high physiological as well as pathological importance. Physiological mechanisms mediated by NO mainly include angiogenesis, growth, puberty and senescence. NO has vital roles in normal reproduction, including steroidogenesis, gametogenesis and the regulation of germ-cell apoptosis. In females, NO stimulates an inflammatory cascade to induce ovulation, decreases steroidogenesis in luteal and granulosa cells, and acts as a paracrine factor to mediate reproductive cycles and implantation. In males, NO is a key player for steroidogenesis, erectile functions, sperm capacitation and acrosome reaction. Moreover, NO is also a regulator of Sertoli cell-germ cell interaction and maintenance of the blood-testis barrier. In pathological conditions such as infections, increased nitric oxide synthase (NOS) activities stimulate the excessive synthesis of NO which acts as a proinflammatory mediator inducing oxidative stress (OS), which is detrimental to reproductive functions in both males and females. During impregnation, the overproduction of NO results in uterine epithelial cell inflammation and immune rejection of implantation. Excessive NO synthesis disrupts gonadal functions, and induces germ cell apoptosis and oxidative damage to the germ cells. This review elucidates how the differences in NO expression levels account for its beneficial and adverse impacts upon male and female fertility.

PITX2 regulates steroidogenesis in granulosa cells of dairy goat by the WNT/β-catenin pathway

Paired-like homeodomain transcription factor 2 (PITX2), a major driver of multiple tissue development, is a transcription factor that regulates gene expression in organisms. However, it is unknown if PITX2 regulates goat granulosa cell (GC) steroidogenesis. Therefore, we investigated the role and mechanism of PITX2 in GC steroidogenesis. In our study, PITX2 significantly facilitated the secretion level of estrogen and progesterone through increasing CYP11A1, CYP19A1, and STAR mRNA and protein expressions in GCs. Furthermore, PITX2 participated in the WNT pathway, enhancing the production of E2 and P4 in GCs. PITX2 in GCs increased the DVL-1 and CTNNB1 expression, involved in the WNT/β-catenin signaling pathway related to steroidogenesis. Moreover, GC steroidogenesis-related gene translation was decreased by CTNNB1-siRNA but enhanced when transfected with PITX2. PITX2 regulates secretion of E2 and P4 from GCs via the WNT/β-catenin pathway and alters GC proliferation and steroidogenesis. These findings will help understand the role of PITX2 in goat ovarian follicular development and oocyte maturation.

Emerging mycotoxins and reproductive effects in animals: A short review.

Emerging Fusarium mycotoxins beauvericin (BEA), enniatins (ENNs), and moniliformin (MON) are gaining increasing interest due to their wide presence especially in cereals and grain-based products. In vitro and in vivo studies indicate that Fusarium mycotoxins can be implicated in reproductive disorders in animals. Of these mycotoxins, BEA may affect reproductive functions, impairing the development of oocytes in pigs and sheep. Studies show dramatic inhibitory effects of BEA and ENNA on bovine granulosa cell steroidogenesis. ENNs also inhibit boar sperm motility and cause detrimental effects on embryos in mice and pigs. Although little data are reported on reproductive effects of MON, in vitro studies show inhibitory effects of MON on Chinese hamster ovary cells. The present review aims to summarize the reproductive toxicological effects of emerging Fusarium mycotoxins BEA, ENNs, and MON on embryo development, ovarian function, and testicular function of animals. In vitro and in vivo toxicological data are reported although additional studies are needed for proper risk assessment.

Cytosine-phosphate-guanine oligodeoxynucleotides regulate the cell cycle, apoptosis, and steroidogenesis of mouse ovarian granulosa cells by targeting inhibin alpha (1 ~ 32) fragments.

Cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODNs), which exist in vertebrate, bacterial, and viral genomes, are regarded as strong immune adjuvants. To date, the biological activities of CpG-ODNs in reproduction remain unknown. Here, we investigated the effects of CpG-ODNs on the cell cycle, apoptosis, and steroidogenesis in mouse granulosa cells (mGCs), in combination with inhibin alpha (1 ~ 32) fragments. mGCs were transfected with pEGFP (containing green fluorescent protein, as a control), pEGISI (containing inhibin alpha (1 ~ 32) fragments), or pEGISI-CpG-ODNs (containing inhibin alpha (1 ~ 32) fragments and CpG-ODNs motifs) plasmid for 48h in vitro. Our results showed that the mRNA and protein expression levels of inhibin alpha were downregulated in mGCs transfected with pEGISI-CpG-ODNs, compared to those transfected with pEGISI. Flow cytometry demonstrated that pEGISI-CpG-ODNs transfection promoted cell proliferation (for example, increasing the number of cells in S and G2 phases) and decreased apoptosis, compared to pEGISI transfection. The present study also indicated that the expression of cell cycle-related genes (cyclin D2, cyclin D3, cyclin E1, Cdk2, and Cdk6) was increased, while the expression of apoptosis-related factors (Fas, FasL, caspase-8, and caspase-3) decreased after pEGISI-CpG-ODNs treatment. Additionally, pEGISI-CpG-ODNs reversed the effect of pEGISI on the secretion of estradiol in mGCs, which was further validated by upregulating the levels of its synthesis-related factors (StAR, Cyp11a1, and 17β-HSD II). Nevertheless, pEGISI-CpG-ODNs or pEGISI did not affect the concentration of progesterone nor changed the expression levels of its synthesis-related factors (3β-HSD I and Cyp19a1). In conclusion, this study demonstrated that CpG-ODNs may affect the cell cycle, apoptosis, and steroidogenesis by targeting the effects of inhibin alpha (1 ~ 32) fragments, supporting the potential role of CpG-ODNs in the development of granulosa cells.

IL-15 Participates in the Pathogenesis of Polycystic Ovary Syndrome by Affecting the Activity of Granulosa Cells.

BackgroundLow-grade chronic inflammation may contribute to the pathogenesis of polycystic ovary syndrome (PCOS). Interleukin-15 (IL-15) is a proinflammatory cytokine involved in the development of chronic inflammation leading to obesity-associated metabolic syndrome. However, the concentration of IL-15 in follicular fluid of patients with PCOS has yet been evaluated.ObjectivesThe aim of this study is to evaluate the expression level of IL-15 in both patients with PCOS and PCOS mice model and investigate the functional effect of IL-15 on ovarian granulosa cells.MethodsThe level of IL-15 in follicular fluid (FF) was measured using cytokine array and enzyme linked immunosorbent assay (ELISA) in two cohorts from 23 PCOS patients and 18 normo-ovulatory controls. PCOS mice model was induced by subcutaneously implanted with letrozole pellet for 21 days. The expression level of IL-15 in serum, ovarian, and subcutaneous adipose tissue in PCOS mice model was measured by ELISA, real-time polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and immunofluorescence. The effect of IL-15 on the proliferation and apoptosis of the KGN cells and mouse ovarian granulosa cells (GCs) were detected by CCK-8 assay and flow cytometry, respectively. Transcript expression of 17α-hydroxylase17,20-lyase (CYP17A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), FSH receptor (FSHR), steroidogenic acute regulatory protein (StAR), and proinflammatory cytokine were quantified using RT-PCR. The protein level and phosphorylation level of p38 MAPK and JNK are detected by Western blot. Concentration of dehydroepiandrosterone sulfate (DHEAS) and progesterone (P)were measured by ELISA.ResultsIL-15 expression in follicular fluid of patients with PCOS was significantly elevated compared with the control group, and similar results were observed in the ovarian and subcutaneous adipose tissue of PCOS mice models. Furthermore, the elevated FF IL-15 levels have a positive correlation with the serum testosterone levels. FSHR co-localized with IL-15 indicating that IL-15 production originate from ovarian granulose cells. IL-15 treatment inhibited proliferation and promoted apoptosis of KGN cells and mouse GCs. Moreover, IL-15 upregulated the transcription levels of CYP17A1, IL-1b and Ifng KGN cells. Similar results were observed in mouse GCs except concentration of DHEAS was higher in IL-15 treatment. IL-15 promoted p38 MAPK and JNK phosphorylation in KGN cells, treating KGN cells with p38 MAPK inhibitor SP600125 and JNK inhibitor SB203580 could reverse the effect of IL-15 on the proliferation and function of KGN cells.ConclusionThe results indicate that IL-15 is involved in the pathogenesis of PCOS potentially by affecting survival, the inflammation state and steroidogenesis of granulosa cells. The practical significance of this association between IL-15 and the pathogenesis of PCOS needs further investigation.

Post-transcriptional silencing of Bos taurus prion family genes and its impact on granulosa cell steroidogenesis

Prion proteins constitute a major public health concern, which has partly overshadowed their physiological roles in several scenarios. Indeed, these proteins were implicated in male fertility but their role in female fertility is relatively less explored. This study was designed to evaluate the role of SPRN and PRNP prion family genes in bovine follicular steroidogenesis pathways. Post-transcriptional SPRN and PRNP silencing with siRNAs was established in bovine granulosa cell (GC) in vitro culture, and gene expression and progesterone and estradiol concentrations were evaluated. SPRN knockdown, led to a downregulation of CYP11A1 mRNA levels (2.1-fold), and PRNP knockdown led to an upregulation of SPRN mRNA levels (2.3-fold). CYP19A1 expression and estradiol synthesis was not detected in any experimental group. Finally, SPRN knockdown led to a mild reduction in progesterone production in GCs and this was the only experimental group that did not exhibit an increment in progesterone levels after 48 h of culture. As a conclusion, it was possible to detect the expression of the SPRN gene in bovine GCs, a potential interaction between SPRN and PRNP regulation, and the impact of SPRN expression on CYP11A1 and progesterone levels. These findings bring new insights into the role of these genes in ovarian steroidogenesis and female reproductive physiology.

Effects of transforming growth factor β1 on steroidogenesis of feline granulosa cells cultured

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. To determine if transforming growth factor β1 (TGFB1), activin, epidermal growth factor (EGF), follicle stimulating hormone (FSH), luteinizing hormone (LH), melatonin, and insulin-like growth factor 1 (IGF1) regulate granulosa cell steroidogenesis and proliferation in cats, three experiments were conducted in winter season. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48h after an initial 48h plating in 10% fetal calf serum. Treatment with IGF1 and FSH increased (P <0.05) estradiol production by 2.3- and 1.33-fold, respectively. In contrast, TGFB1 blocked (P <0.05) IGF1-induced estradiol production and inhibited FSH-induced estradiol production by 60%. Combined with FSH or FSH plus IGF1, TGFB1 inhibited (P <0.05) cell proliferation, whereas TGFB1 increased progesterone production by 2.8-fold in the presence of FSH plus IGF1. EGF decreased (P <0.05) FSH plus IGF1-induced estradiol production by 89% but did not affect progesterone production or cell numbers. Activin did not affect (P >0.10) cell numbers or steroidogenesis in the presence of FSH plus IGF1. Melatonin and LH decreased (P <0.05) estradiol production 53% and 59%, respectively, without affecting progesterone production or cell proliferation. The present study has identified TGFB1 as a major regulator of feline ovarian function, in addition to EGF, IGF1, melatonin, LH and FSH. These studies will provide useful information for future development of fertility control in feline species.

Silver nanoparticles induce oxidative stress, apoptosis and impaired steroidogenesis in ovarian granulosa cells of cattle

There have been increased effects of silver nanoparticle (Ag-NPs) on livestock during the past decade, but data related to adverse effects of Ag-NPs on reproductive tissues are limited. In the present study, the possible cytotoxic effects of Ag-NPs on oxidant/antioxidant balance, apoptosis and steroid hormone production in ovarian granulosa cells of cattle were studied.Cultured granulosa cells were treated with 10 nm Ag-NPs at various concentrations (1–100 µg/ml) for 24 h, and cell toxicity, oxidant/antioxidant markers, reactive oxygen species (ROS) production, abundances of apoptotic/antiapoptotic and steroidogenesis related mRNA transcripts were determined. The amount of DNA fragmentation was also determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results indicated treatment of granulosa cells with Ag-NPs induced an increase of reactive oxygen species production concomitant with increased malondialdehyde concentrations and decreased antioxidant enzyme activities. There was a maximal percentage of TUNEL+ cells (46.6%) after treatment with 50 and 100 µg/ml of Ag-NPs. The Ag-NPs could induce apoptosis in granulosa cells as indicated by the increase in caspase-3 activity, and larger abundance of BCL2 associated X (BAX) and lesser abundance of B-cell lymphoma 2 (BCL2) mRNA transcripts. The abundance of steroidogenic enzyme mRNA transcripts decreased concomitant with suppression of steroid hormone synthesis from Ag-NP-treated cells. Findings indicate silver nanoparticles (SNP) induce apoptosis and oxidative stress and change the pattern of steroid hormone synthesis in granulosa cells of cattle. The results indicate Ag-NPs may inhibit the function and viability of ovarian cells.

Copper exposure disrupts ovarian steroidogenesis in human ovarian granulosa cells via the FSHR/CYP19A1 pathway and alters methylation patterns on the SF-1 gene promoter

Information on the effects of copper on reproduction is limited. Our previous study indicated that copper induces abnormal steroidogenesis in human ovarian granulosa cells, but the underlying mechanism remains unclear. In this study, human ovarian granulosa cells were treated with multiple concentrations of copper for 24 h. After treatment, the 17-estradiol levels were significantly increased (29.83 % and 45.12 %, respectively) in the 1.0 and 2.0 μg/mL groups but decreased (23.06 % and 31.56 %, respectively) in the 20.0 and 40.0 μg/mL groups (P < 0.05). Similar changes in the levels of FSHR, StAR, CYP11A1, CYP19A1, HSD3β1, and SF-1 were observed. The protein levels of FSHR were increased in the 2.0 μg/mL group but decreased in the 20.0 and 40.0 μg/mL groups (P < 0.05). Moreover, copper partially reversed the FSH-induced increase in FSHR, CYP19A1 and 17-estradiol levels, and the decreased effect of the FSH receptor binding inhibitor fragment on FSHR, CYP19A1, and 17-estradiol became more apparent after adding copper. Additionally, the total methylation levels of the SF-1 promoter and DNMTs expression were significantly decreased following copper treatment. Overall, our results indicate that copper exposure induces steroidogenesis disorders via the FSHR/CYP19A1 pathway and changes DNA methylation on the SF-1 promoter in human ovarian granulosa cells.

Vitamin D deficiency inhibits microRNA-196b-5p which regulates ovarian granulosa cell hormone synthesis, proliferation, and apoptosis by targeting RDX and LRRC17.

BackgroundIn polycystic ovary syndrome (PCOS), ovarian physiology is tightly linked to the metabolic disturbances observed in this disease. Vitamin D (VD) plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells (GCs). However, its role in the proliferation and apoptosis of ovarian GCs is unclear. The present study aimed to investigate the role of microRNA-196-5p (miR-196b-5p) in the hormone synthesis, proliferation, and apoptosis of ovarian GCs.MethodsThe abnormal expression of miRNAs in ovarian tissues of VD-deficient mice was analyzed using transcriptome sequencing. The direct target of miR-196b-5p was predict and confirmed by bioinformatics analysis and the dual-luciferase reporter assay. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect the levels of miR-196b-5p, cell proliferation was detected via the CCK8 assay, and cell apoptosis and reactive oxygen species (ROS) were measured via flow cytometry. The levels of radixin (RDX), leucine rich repeat containing 17 (LRRC17), aromatase (CYP19A1), and glucose transporter 4 (GLUT4) were detected by performing RT-qPCR or western blot.ResultsWe found that miR-196b-5p was significantly downregulated among the 672 miRNAs that were differentially expressed (DE) in VD-deficient mice. In addition, the results demonstrated that downregulated expression of miR-196b-5p significantly increased the level of RDX and LRRC17, and reduced expression of miR-196b-5p significantly promoted ovarian GC apoptosis and inhibited cell proliferation. Downregulated expression of miR-196b-5p promoted cellular ROS production and inhibited sex hormone production and glucose uptake. Transfection with miR-196b-5p mimics significantly increased the expression of CYP19A1 and GLUT4 and decreased the RDX and LRRC17 levels in ovarian GCs.ConclusionsThis study shows that miR-196b-5p can regulate the oxidative stress (OS), glucose uptake, and steroid production pathway of GCs, thus promoting follicular development and maturation. This is a step towards a feasible treatment for PCOS.

Effects of bone morphogenetic protein 4, gremlin, and connective tissue growth factor on estradiol and progesterone production by bovine granulosa cells.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin [GREM]) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GCs and its interaction with GREM or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GCs, but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GCs. In small-follicle GCs, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but GREM inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GCs. In large-follicle GCs, BMP4 (10 to 30 ng/mL) increased (P < 0.05) GC numbers and GREM (100 ng/mL) blocked this effect. In large-follicle GCs, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, GREM, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large vs. small follicles.

PSXII-23 Sirtuins expression and activity in bovine granulosa cells are regulated by hormones that influence ovarian steroidogenesis

Abstract Sirtuins (SIRTs) are a family of seven NAD+-dependent histone deacetylases that regulate several biological reactions. How SIRTs regulate ovarian steroidogenesis in cattle remains to be fully unveiled. We hypothesize that SIRTs expression and activity are regulated by hormones that influence steroidogenesis in bovine granulosa cells (GC). Bovine ovaries were collected at an USDA-inspected commercial slaughterhouse and GC were isolated from small antral follicles (1–5 mm on surface diameter). Cells were treated with hormones that regulate ovarian folliculogenesis: FSH, IGF1, fibroblast growth factor (FGF) 2, FGF9, and their combinations. Cells were cultured for 24h for total RNA isolation (n = 6 pools) with miRNeasy microkit (Qiagen) or for 48h for isolation of nuclear and cytoplasmic extracts (n = 3 pools) with EpiQuik Nuclear Extraction Kit (Epigentek) according to the manufacturers’ instructions. Relative mRNA abundance was quantified via qPCR and expressed as 2-ΔΔCt using the relative comparative threshold cycle (Ct) whereas SIRTs activity in nuclear (SIRTs 1, 6, and 7) and cytoplasmic (SIRTs 2, 4, and 5) extracts was analyzed with the Epigenase Universal SIRT Activity/Inhibition assay kit (Epigentek) following the manufacturer’s instructions. Data were analyzed via ANOVA with GLM procedures of SAS for Windows. In terms of mRNA relative abundance, FSH+IGF1+IGF9 increased mRNA relative expression of SIRTs 2 to 7 in comparison to negative control and of SIRTs 2, 3, 4, 6, and 7 in comparison to FSH+IGF1; FSH+IGF1+IGF2 increased mRNA relative abundance of SIRTs 2 and 6 in comparison to FSH+IGF1; FGF2 alone increased SIRT1 in comparison to negative control (P &amp;lt; 0.05). In term of SIRTs activity, FGF2 alone increased nuclear SIRTs activity in comparison to FSH, IGF1, FSH+IGF1, and FGF9 alone; FSH+IGF1+IGF2 increased cytoplasmic SIRTs activity in comparison to all treatments (P &amp;lt; 0.05). Taken together, our data demonstrate that SIRTs expression and activity in bovine GC are regulated by hormones that influence steroidogenesis.

FSH stimulated bovine granulosa cell steroidogenesis involves both canonical and noncanonical WNT signaling

Gonadotrophins play key roles in follicular development; however, the underlying molecular mechanisms are not fully understood. Follicle stimulating hormone (FSH) regulation of aromatase and subsequent estradiol (E2) production relies on β-catenin, a key effector of WNT signaling. We previously demonstrated that treatment with the canonical WNT inhibitor, IWR-1, reduced FSH induced bovine granulosa cell E2 production in vitro. Here we demonstrated that intrafollicular injection in vivo with IWR-1 alters steroidogenesis and triggers a significant decrease in estrogen to progesterone ratio in the IWR-1 treated follicles compared to diluent injected control follicles. We next examined markers of canonical and noncanonical WNT signaling in dominant and subordinate follicles collected at different stages of follicular development and showed that protein for both CTNNB1 (canonical pathway) and phosphorylated (p)-LEF1 (noncanonical pathway) was significantly elevated in dominant compared to subordinate follicles at the early dominance stage of development. Therefore, we hypothesized that canonical and/or noncanonical WNT ligands modulate FSH stimulated E2 production. Hence, we examined the effects of specific WNT ligands on FSH stimulated E2 production in the absence of endogenous WNT production in vitro. Universal WNT signaling inhibitor, LGK-974 was able to inhibit FSH stimulation of E2 and reduce the abundance of proteins linked to canonical and noncanonical WNT pathway activation. Supplementation with the canonical ligand WNT2b did not affect the inhibitory effects of LGK-974 on FSH stimulated E2 production but rescued the LGK-974 mediated inhibition of CTNNB1 (canonical pathway) but not p-LEF1, p-JNK or p-P38 abundance (noncanonical pathway) abundance. In contrast, WNT5a treatment rescued FSH stimulated estradiol production and indices of activation of both the canonical (CTNNB1) and noncanonical (p-LEF1, p-JNK and p-P38) WNT signaling pathways in LGK-974 treated granulosa cells. Taken together, these results suggest that both canonical and noncanonical WNT pathways activation is linked to FSH stimulation of E2 production by bovine granulosa cells.

Novel insights into the regulation of LATS2 kinase in prehierarchical follicle development via the Hippo pathway in hen ovary

The large tumor suppressor homolog 2 (LATS2), one of the central regulators of the Hippo/MST signaling pathway, plays an inhibitory role in ovarian function and different organ development and growth in mammals. However, the exact roles and molecular regulatory mechanisms of LATS2 in chicken granulosa cell (GC) proliferation, differentiation, and steroidogenesis required for ovarian follicle growth, development, and follicular selection remain poorly understood. This study demonstrated that the LATS2 protein was predominantly localized in the oocytes and undifferentiated GCs of various-sized prehierarchical follicles of the hen ovary. Expression levels of LATS2 mRNA were significantly higher in the smaller follicles (from 1 mm to 5.9 mm in diameter) and the GCs than in the larger follicles (6–6.9 mm in diameter up to F1). Moreover, we found that high levels of LATS2 suppressed the GC proliferation and the mRNA and protein expression of the genes serving as the biomarkers of follicle selection, GC differentiation, and steroidogenesis in the GCs, including FSHR, STAR, CYP11A1, ESR1, and ESR2. Interestingly, the LATS2 significantly downregulated SAV1 and YAP1 transcripts but upregulated the expression of STK3, STK4, TEAD1, and TEAD3 mRNA. Our study provided evidences that STK3/4-LATS2-YAP1 not only acts as a suppressor of cell proliferation and follicle selection but also LATS2 may serve as an enhancer in cell proliferation and follicle selection through the YAP1-LATS2 and the LATS2-STK3/4 feedback loops by promoting the expression of TEAD1/3 but inhibiting the expression of SAV1 transcripts in the prehierarchical follicle development of hen ovary. Taken together, the present study initially revealed the pivotal role and molecular mechanism of LATS2 in the regulation of hen prehierarchical follicle development by controlling GC proliferation, differentiation, steroidogenesis, and follicle selection via the Hippo/MST signaling pathway.

Epigallocatechin-3-gallate stimulates StAR expression and progesterone production in human granulosa cells through the 67-kDa laminin receptor-mediated CREB signaling pathway.

Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechins extracted from green tea. The health benefits of EGCG have been extendedly studied. Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function. Granulosa cells in the ovary are essential for steroid hormone production. To date, the effect of EGCG on steroidogenesis in human granulosa cells remains unclear. In the present study, we examine the physiological concentrations of EGCG on steroidogenesis in a steroidogenic human granulosa-like tumor cell line, KGN. Our results demonstrate that treatment with EGCG upregulates steroidogenic acute regulatory protein (StAR) expression and increases progesterone (P4) production. EGCG does not affect the expression levels of other steroidogenesis-related enzymes, such as P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. In addition, we identify the expression of 67-kDa laminin receptor (67LR) in KGN cells. Moreover, EGCG-induced StAR expression and P4 production require the 67LR-mediated activation of the PKA-CREB signaling pathway. These results provide a better understanding of the function of EGCG on ovarian steroidogenesis, which may lead to the development of alternative therapeutic approaches for reproductive disorders.

MiR-145 regulates steroidogenesis in mouse primary granulosa cells through targeting Crkl

AimsIt has been demonstrated that miR-145 is expressed in primordial follicles and modulates the initiation of primordial follicle development. We aimed to explore the function of miR-145 in mouse granulosa cells (mGCs). Materials and methodsThe proliferation and differentiation of GCs were examined via MTT, EDU assay, QRT-PCR, ELISA and electron microscope analysis. The target of miR-145 was determined by bioinformatics analysis and luciferase reporter assay and the molecular mechanisms were examined via western blot and quantitative Real-Time RT-PCR. Key findingsWe proved that down-regulation of miR-145 could inhibit GCs proliferation and differentiation. In addition, we provided evidence that Crkl was the target gene of miR-145. The miR-145 antagomir caused an increase in Crkl expression and activation of the JNK/p38 MAPK pathway. Overexpression of Crkl with pEGFP-N1-Crkl vector inhibited GCs differentiation and progesterone synthesis as well as activation of the JNK/p38 MAPK pathway. SignificanceOur study shows that miR-145 targets Crkl and through the JNK/p38 MAPK signaling pathway promotes the GCs proliferation, differentiation, and steroidogenesis. MiR-145 may play an important role in the ovarian physiology and pathology.

Effects of Orexin B on Swine Granulosa and Endothelial Cells.

Simple SummaryThe follicle is the ovarian functional unit. It is mainly composed of granulosa cells and angiogenesis is crucial to guarantee its development till ovulation. Carrying on our previous studies on the orexin system in the ovary, we presently demonstrate a potential role of orexin B in the control of granulosa cells’ oxidative stress and of the angiogenesis event.In addition to the well-known central modulatory role of orexins, we recently demonstrated a peripheral involvement in swine granulosa cells for orexin A and in adipose tissue for orexin B (OXB). The aim of present research was to verify immunolocalization of OXB and its potential role in modulating the main features of swine granulosa cells. In particular, we explored the effects on granulosa cell proliferation (through the incorporation of bromodeoxyuridine), cell metabolic activity (as indirect evaluation by the assessment of ATP), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test and the non-enzymatic reducing power by FRAP test). Our data point out that OXB does not modify granulosa cell growth, steroidogenesis and superoxide anion generation. On the contrary, the peptide stimulates (p < 0.05) nitric oxide output and non-enzymatic reducing power. Since new vessel growth is crucial for ovarian follicle development, a further aim of this study was to explore the expression of prepro-orexin and the effects of OXB on swine aortic endothelial cells. We found that the peptide is ineffective in modulating cell growth, while it inhibits redox status parameters. In addition, we demonstrated a stimulatory effect on angiogenesis evaluated in fibrin gel angiogenesis assay. Taken together, OXB appears to be potentially involved in the modulation of redox status in granulosa and endothelial cells and we could argue an involvement of the peptide in the follicular angiogenic events.