Bovine Granulosa Research Articles

WITHDRAWN:Estrogen receptor β (ERβ) induces bovine ovarian granulosa cell (BGC) autophagy via the AKT/mTOR pathway.

Ahead of Print article withdrawn by Editorial Board.

Unraveling proteome changes and potential regulatory proteins of bovine follicular Granulosa cells by mass spectrometry and multi-omics analysis

BackgroundIn previous study, we performed next-gene sequencing to investigate the differentially expressed transcripts of bovine follicular granulosa cells (GCs) at dominant follicle (DF) and subordinate follicle (SF) stages during first follicular wave. Present study is designed to further identify the key regulatory proteins and signaling pathways associated with follicular development using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multi-omics data analysis approach.MethodsDF and SF from three cattle were collected by daily ultrasonography. The GCs were isolated from each follicle, total proteins were digested by trypsin, and then proteomic analyzed via LC-MS/MS, respectively. Proteins identified were retrieved from Uniprot-COW fasta database, and differentially expressed proteins were used to functional enrichment and KEGG pathway analysis. Proteome data and transcriptome data obtained from previous studies were integrated.ResultsTotal 3409 proteins were identified from 30,321 peptides (FDR ≤0.01) obtained from LC-MS/MS analysis and 259 of them were found to be differentially expressed at different stage of follicular development (fold Change > 2, P < 0.05). KEGG pathway analysis of proteome data revealed important signaling pathways associated with follicular development, multi-omics data analysis results showed 13 proteins were identified as being differentially expressed in DF versus SF.ConclusionsThis study represents the first investigation of transcriptome and proteome of bovine follicles and offers essential information for future investigation of DF and SF in cattle. It also will enrich the theory of animal follicular development.

Oncostatin M and its receptors mRNA regulation in bovine granulosa and luteal cells

Oncostatin M (OSM) and its receptor (OSMR) are members of the interleukin-6 family cytokines. Although OSM and OSMR expression was detected in human ovaries, their function and regulation during follicle development, ovulation and luteolysis have not been studied in any species. The aim of the present study was to investigate the levels of OSM and OSMR mRNA in bovine ovaries and the effect of OSM treatment on cultured granulosa cells. OSM mRNA was not detected in granulosa cells obtained from follicles around the time of follicular deviation and from pre-ovulatory follicles, whereas OSMR transcript levels were greater in granulosa cells of atretic subordinate follicles (P < 0.001). Abundance of OSMR mRNA increased in granulosa cells of preovulatory follicles, collected at 12 and 24 h after the ovulatory stimulus with gonadotropins (P < 0.001). In the luteal tissue, OSM mRNA abundance levels were higher at 24–48 h after PGF-induced luteolysis (P < 0.01) compared to 0 h, whereas OSMR mRNA was transiently increased at 2 h after PGF treatment (P < 0.05). In cultured granulosa cells, 10 ng/mL OSM in the presence of FSH increased BAX/BCL2 mRNA ratio (P < 0.05) compared to the control. Moreover, 100 ng/mL OSM in the presence of FSH increased OSMR (P < 0.05) and decreased XIAP mRNA (P < 0.05) levels, compared to the control group. These findings provide the first evidence that OSMR is regulated during follicle atresia, ovulation and luteolysis, and that OSM from other cells may mediate granulosa and luteal cell function, regulating the expression of genes involved in cell's viability.

MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation

BackgroundOocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. With respect to this, previously, we identified altered expression of microRNA-130b (miR-130b) during oocyte maturation. Here, we aimed to investigate the role of miR-130b in bovine granulosa and cumulus cell function, oocyte maturation and preimplantation embryo development using gain- and loss-of- function approach.MethodsFor this study, the granulosa cells, cumulus cells and the oocytes were collected from ovaries obtained from slaughterhouse. The genes targeted by miR-130b were identified using dual-luciferase reporter assay. The role of miR-130b in granulosa and cumulus cell function was investigated by increasing and inhibiting its expression in in vitro cultured cells using miR-130b precursor and inhibitor, respectively while the role of miR-130b on oocyte development, immature oocytes were microinjected with miR-130b precursor and inhibitor and the polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial were determined in each oocyte group 22 h after microinjection. Moreover, to investigate the role of miR-130b during preimplantation embryo development, zygote stage embryos were microinjected with miR-130b precursor or inhibitor and the cleavage rate, morula and blastocyst formation was analyzed in embryos derived from each zygote group after in vitro culture.ResultsThe luciferase assay showed that SMAD5 and MSK1 genes were identified as the direct targets of miR-130b. Overexpression of miR-130b increased the granulosa and cumulus cell proliferation, while inhibition showed the opposite phenotype. Apart from these, modulation of miR-130b altered the lactate production and cholesterol biosynthesis in cumulus cells. Furthermore, inhibition of miR-130b expression during oocyte in vitro maturation reduced the first polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial activity, while inhibition of miR-130b during preimplantation embryo development significantly reduced morula and blastocyst formation.ConclusionThis study demonstrated that in vitro functional modulation of miR-130b affected granulosa and cumulus cell proliferation and survival, oocyte maturation, morula and blastocyst formation suggesting that miR-130b is involved in bovine oocyte maturation and preimplantation embryo development.

Effects of the Pro-Inflammatory Cytokine Interleukin6 on Steroidogenesis by Bovine Granulosa (GC) and Theca (TC) Cells

Effects of the Pro-Inflammatory Cytokine Interleukin6 on Steroidogenesis by Bovine Granulosa (GC) and Theca (TC) Cells

0214 FSH dependent and IGF-1 independent phosphorylation of β-catenin is similar in bovine and human granulosa cells

0214 FSH dependent and IGF-1 independent phosphorylation of β-catenin is similar in bovine and human granulosa cells

Direct effects of the algal toxin, domoic acid, on ovarian function: Bovine granulosa and theca cells as an in vitro model

Domoic acid (DA) is a potent neurotoxin produced by alga Pseudo-nitzschia spp. and has been associated with reproductive disorders in mammals. The aim of this study was to investigate if DA can affect the reproductive system via direct action on ovarian function. Bovine granulosa and theca cells were used as in vitro models for evaluating DA effects on ovarian cell proliferation and steroid production. In small-follicle granulosa cells (SMGC), cell proliferation and estradiol (E2) production was not affected (P>0.05) while progesterone (P4) production was inhibited (P<0.05) by DA at all doses tested. In large-follicle granulosa cells (LGGC), DA had no effect (P>0.05) on cell proliferation or P4 production while E2 production was stimulated by 1 and 5µg/ml DA (P<0.05). DA (1µg/ml) attenuated (P<0.05) insulin-like growth factor 1 (IGF-1)-induced P4 production by large-follicle theca cells (LGTC), but did not affect androstenedione (A4) production or proliferation of LGTC. In glutamate-free medium, DA inhibited (P<0.05) SMGC E2 production and this inhibition was similar to inhibition of E2 by trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid monohydrate (ACPD; a selective metabotropic glutamate receptor subtype agonist) while kainic acid (KA; an ionotropic glutamate receptor subtype agonist) had no effect (P>0.10) on E2 production. Collectively, these results show for the first time that DA has direct effects on ovarian GC and TC steroidogenesis. Because DA inhibited E2 and P4 production, DA has the potential to be an endocrine disruptor.

The effect of PCB126, 77, and 153 on the intracellular mobilization of Ca+2 in bovine granulosa and luteal cells after FSH and LH surge in vitro

Polychlorinated biphenyls (PCBs) are a group of persistent environmental pollutants that impair cattle reproduction. Among other effects, PCBs can disturb the intracellular mobilization of Ca(+2) in several cell types. Hence, it is possible that they disrupt the transduction of intracellular signals generated from gonadotropin (FSH/LH) receptors. In steroidogenic ovarian cells, a defect in Ca(+2) mobilization may have a detrimental influence on two important processes: the secretion of steroids (E2 or/and P4) and their morphological and functional differentiation. The aim of this study was to determine the influence of PCBs: 126 (dioxin-like) 77 (ambivalent) and 153 (estrogen-like) and a mixture of PCBs (Aroclor 1248) on these processes. Bovine granulosa and luteal cells were incubated for 72 hrs with PCBs (100 ng/ml), followed by Fura 2AM dye, and the fluctuations in intracellular Ca(+2) mobilization after FSH/LH treatment were determined using an inverted microscope coupled with a CCD camera. The intensity and area of fluorescence excited by UV light were detected in the green spectrum of visible light. Aroclor 1248 and PCBs 153 and 77 significantly decreased (P < 0.01-0.001) the effect of FSH on intracellular Ca(+2) mobilization in granulosa cells. In luteal cells, the most effective PCB on this process was PCB 77. The results revealed adverse effects of PCBs on the mobilization of intracellular Ca(+2). Moreover, the estrogen-like congeners were found to more effectively disturb this process than the dioxin-like PCB 126. Hence, it is possible for PCBs to have a negative influence on reproductive processes by affecting calcium mobilization.

In vitro effects of deoxynivalenol and β-Zearalenol alone and in combination on steroidogenesis in bovine small granulosa cells

In vitro effects of deoxynivalenol and β-Zearalenol alone and in combination on steroidogenesis in bovine small granulosa cells

Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-α-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells

Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary

The Hedgehog System in Ovarian Follicles of Cattle Selected for Twin Ovulations and Births: Evidence of a Link Between the IGF and Hedgehog Systems1

Hedgehog signaling is involved in regulation of ovarian function in Drosophila, but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or theca (TC) cells of small (1-5 mm) antral follicles by in situ hybridization and of larger (5-17 mm) antral follicles by real-time RT-PCR from ovaries of cyclic cows genetically selected (Twinner) or not selected (control) for twin ovulations. Expression of IHH mRNA was localized to GC and cumulus cells, whereas PTCH1 mRNA was greater in TC than in GC. Estrogen-active (E-A; follicular fluid concentration of estradiol > progesterone) versus estrogen-inactive follicles had a greater abundance of mRNA for IHH in GC and PTCH1 in TC. Abundance of IHH mRNA in GC was not affected by cow genotype, whereas TC PTCH1 mRNA was less in large E-A follicles of Twinners than in controls. In vitro, estradiol and wingless-type (WNT) 3A increased IHH mRNA in IGF1-treated GC. IGF1 and BMP4 treatments decreased PTCH1 mRNA in small TC. Estradiol and LH increased PTCH1 mRNA in IGF1-treated TC from large and small follicles, respectively. In summary, functional status of ovarian follicles was associated with differences in hedgehog signaling in GC and TC. We hypothesize that as follicles grow and develop, increased free IGF1 may suppress expression of IHH mRNA by GC and PTCH1 mRNA by TC, and these effects are regulated in a paracrine way by estradiol and other intra- and extragonadal factors.

In Vivo Expression of Cysteine-Rich 61 and Angiogenic Regulators in Bovine Ovarian Follicles.

We have previously shown that the angiogenic inducer, cysteine-rich, angiogenic inducer, 61 (CYR61), is highly expressed in the day 4 bovine corpus luteum (CL). Along with some of the known angiogenic regulators, vascular endothelial growth factor A (VEGF), fibroblast growth factor 2 (FGF2) and matrix metalloproteinase 2 (MMP2), CYR61 may function to promote the high rate of angiogenesis that occurs during the early development of the CL. Since the folliculo-luteal transition results in the infiltration of blood vessels from the vascularized theca interna into the avascular granulosa compartment, we hypothesized that CYR61 may play a critical role in switching on this angiogenic process. However, because it is not known whether ovarian follicles or the surrounding stroma may also synthesize CYR61, our objectives were to determine the expression of CYR61 in bovine granulosa and theca, and relate its expression to know angiogenic regulators such as VEGF, FGF2 and MMP2. Ovaries were removed from midcycle Holstein cows at time 0 (control, no treatment) and at 24 hours or 48 hours following prostaglandin F2 alpha (PGFa) injection. Dissected follicles were measured (diameter; mm); follicular fluid was aspirated; and the granulosa cells and theca interna were separated before RNA was extracted from them. cDNAs generated from granulosa cells and theca interna were analyzed by quantitative PCR to determine the levels of known angiogenic factors, including CYR61, VEGF, FGF and MMP2. Additionally, follicular fluid was analyzed for expression and activity of MMP2 and MMP9 by zymography. Follicular fluid estradiol (E2) and progesterone (P4) concentrations were determined by radioimmunoassay to assess the health status of follicles. The E2 concentrations in the follicular fluid obtained from the largest follicles (diameter 18.9 mm to 22.4 mm) were high, indicating that they were healthy. In addition, plasma P4 concentrations decreased accordingly following PGF2a injection. Further, although there were no differences in mRNA expression of selected angiogenic factors between follicles of PGF2a treated and control cows for any of the endpoints, the mRNA expression of CYR61, FGF2 and MMP2 was always higher (p<0.05) in the thecal than in the granulosa compartment within controls and within each treatment group. In contrast, the mRNA expression of VEGF in the thecal compartment was always lower (p<0.05) than in the hypoxic, granulosa compartment within controls and each treatment group. In summary, CYR61 is reported, for the first time, to be expressed in vivo by granulosa cells and theca interna of bovine ovarian follicles. The function of CYR61 and other angiogenic regulators in the folliculo-luteal transition remains to be elucidated, awaiting analysis of follicles collected at earlier time points, e.g. at 1, 6 and 12 hrs following PGFa injection. This research was supported in part by: NIH grant P01 CA045548 to MAM and by the Multistate Northeast Regional Project NE-1027 to PCWT. (poster)

Thyroglobulin gene is associated with premature ovarian failure

Variants of the thyroglobulin gene were significantly associated with premature ovarian failure in a Korean population. Three single nucleotide polymorphisms and one haplotype were found to be associated with a significant increase in the risk for premature ovarian failure.

Stage-Specific Responses in Gene Expression and Signaling Pathway Activation after Treatment with Prostaglandin F2 Alpha (PGF) and Interferon-Gamma (IFNG) in Bovine Corpus Luteum (CL) and Luteinizing Granulosa Cells.

Stage-Specific Responses in Gene Expression and Signaling Pathway Activation after Treatment with Prostaglandin F2 Alpha (PGF) and Interferon-Gamma (IFNG) in Bovine Corpus Luteum (CL) and Luteinizing Granulosa Cells.

FGFs Differentially Regulate Sprouty Expression and ERK Phosphorylation in Bovine Ovarian Granulosa Cells.

FGFs Differentially Regulate Sprouty Expression and ERK Phosphorylation in Bovine Ovarian Granulosa Cells.

The influence of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and its metabolite—dichlorodiphenyldichloroethylene (DDE) on mRNA expression for NP-I/OT and PGA, involved in oxytocin synthesis in bovine granulosa and luteal cells

The effect of polychlorinated biphenyls (PCBs) congeners (PCB 77, PCB 126, PCB 153) and their technical mixture—Aroclor (Ar) 1248, as well as dichlorodiphenyltrichloroethane (DDT) and its metabolite—dichlorodiphenyldichloroethylene (DDE; two individual isomers p,p′- and o,p′- or their mixture, 95% and 5%, respectively) at the dose of 10 ng/ml each, on the gene expression of (a) oxytocin (OT) precursor—neurophysin–oxytocin (NP-I/OT) and (b) peptidyl glycine-α-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of OT synthesis, was studied. Granulosa cells from follicles >1 cm in diameter, collected on days 19–21 of estrous cycle, and luteal cells from corpora lutea (CL) collected on days 8–12 of the estrous cycle were used. The cells were incubated (6 h) with these xenobiotics and the expression of NP-I/OT and PGA genes was determined. All PCBs increased ( P < 0.05) NP-I/OT gene expression in granulosa cells. Similarly, all PCBs but PCB 126 increased ( P < 0.05) PGA gene expression in these cells. DDT and DDE increased ( P < 0.05) gene expression of NP-I/OT in granulosa cells, while gene expression of PGA in these cells was stimulated ( P < 0.05) by DDE only. The mRNA expression for NP-I/OT and PGA in luteal cells was increased ( P < 0.05) by PCB 77 and PCB 153. Both DDE isomers and mixture also stimulated ( P < 0.05) of NP-I/OT mRNA expression, while increase ( P < 0.05) of PGA mRNA expression was elicited by incubation of these cells with DDE mixture and Ar 1248. Obtained data suggest that PCBs, DDT and DDE can affect the mRNA expression for NP-I/OT and PGA in bovine granulosa and luteal cells.

Effect of polychlorinated biphenyls on the secretion of oxytocin from luteal and granulosa cells in cow: possible involvement of glucocorticoid receptors

Polychlorinated biphenyls (PCBs) stimulate oxytocin secretion from bovine granulosa and luteal cells. Since oxytocin on the one hand is released from ovarian cells by cortisol and on the other hand PCBs can be bound by glucocorticoid receptors (GCr), we have tested the hypothesis that PCBs acting via GCr can stimulate oxytocin secretion. In preliminary studies the effect of RU486 (GCr blocker) on cells viability was tested. Thereafter, a selected dose of RU486 (10&lt;sup&gt;5&lt;/sup&gt;M), which did not affect cell viability, was used in further experiments. It appears that this dose of RU486 completely blocked GCr against cortisol-stimulated oxytocin secretion, in both types of cells. Furthermore, granulosa cells (10&lt;sup&gt;5&lt;/sup&gt;) from follicles of two sizes (&amp;gt;1 cm &amp;lt; in diameter) and luteal cells (10&lt;sup&gt;5&lt;/sup&gt;) from day 5&amp;ndash;10 of estrous cycle were incubated for 72 h with congeners of PCB (126, 77 or 153) at doses of 1, 10 or 100&amp;nbsp;ng/ml each, separately or jointly with RU486. The effect of PCB 77 and 153 on oxytocin secretion was blocked by RU486, but it did not change the effect evoked by PCB126 in both granulosa and luteal cells. We assume that some PCB congeners can affect oxytocin secretion from granulosa and luteal cells acting via GCr.

Adenylyl cyclases in oocyte maturation: A characterization of AC isoforms in bovine cumulus cells

Mammalian oocytes are arrested at the G(2)/M transition in the meiotic cell cycle. It is well known that a decrease in intraoocyte cAMP concentrations accompanies resumption of meiosis, but the precise trigger of this decrease remains a mystery. Follicular somatic cells are intimately coupled to the oocyte and are thought to transmit maturation signals to the oocyte in response to hormonal stimulation. Here, we investigate the nature of the follicular somatic cell response to hormonal stimulation by identifying and characterizing the adenylate cyclase isoforms present in bovine cumulus cells. RT-PCR and Western blot analysis revealed the presence of multiple adenylyl cyclase isoforms in bovine granulosa and cumulus cells. Pharmacological manipulation of the AC isoforms showed that multiple isoforms were indeed active. Our data indicate that the PKC inhibited adenylate cyclases IV and VI and the calcium-stimulated isoform I predominate in bovine cumulus cells.

Bovine mural granulosa cells, and not the oocyte, are the major source of proteases capable of IGFBP-2 degradation

Oocytes can regulate their own development, hence studies identifying and characterising oocyte-secreted factors are crucial to resolving the mechanisms by which oocytes can orchestrate follicular development. The insulin-like growth factor (IGF) system plays an important role during the development of a follicle; however, the regulation of IGF bioavailability is crucial throughout follicular growth. Proteolytic cleavage of the IGF/IGF binding protein (IGFBP) complex increases IGF bioavailability, hence, the production of IGFBP proteases by the oocyte and/or granulosa cells would provide a regulatory mechanism whereby they could regulate their own exposure to IGFs. The present study revealed mural granulosa cells (MGC), and not the oocyte, are the major source of proteases capable of cleaving IGFBP-2 in the developing bovine antral follicle. The addition of recombinant IGF-I or FSH had no effect in terms of modulating IGFBP-2 degradation. This work further supports the existence of a local regulatory mechanism modulating IGF bioavailability.

Effects of β‐OH Butyrate on Bovine Granulosa and Theca Cell Function in Vitro

In this study, the effects of beta-OH butyrate (BHB) levels, associated with a negative energy balance, on bovine granulosa and theca cell function were investigated in vitro. Granulosa and theca cells of healthy large follicles (>8 mm), obtained from slaughterhouse ovaries, were cultured in serum free medium containing 0, 0.5, 1 or 1.5 mm BHB and 3 mm glucose, to mimic the situation in the early postpartum dairy cow. Hormone concentrations (progesterone, oestradiol-17beta and/or androstenedione) in spent medium and cell numbers were measured after 48 h of culture. No effects of BHB on theca cell numbers or on steroid production were observed. In granulosa cells, all BHB treatments evenly increased cell numbers (p < 0.05), while they reduced progesterone and oestradiol-17beta production per cell (p < 0.05). These effects may be attributed to the use of BHB as energy source which is however differently metabolized than glucose. Conclusively, in the presence of physiological glucose concentrations BHB can modulate granulosa but not theca cell function in vitro.