Gremlin, noggin, chordin and follistatin differentially modulate BMP induced suppression of androgen secretion by bovine ovarian theca cells

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle function and steroidogenesis but information is lacking regarding the regulation of BMP signalling by extracellular binding proteins co-expressed in the ovary. In this study we compared the abilities of four BMP binding proteins (gremlin, noggin, chordin, follistatin) to antagonize the action of four different BMPs (BMP2 BMP4, BMP6, BMP7) on LH-induced androstenedione secretion by bovine theca cells in primary culture. Expression of the four BMP binding proteins and BMPs investigated here has previously been documented in bovine follicles. All four BMPs suppressed androstenedione secretion by >85%. Co-treatment with gremlin antagonized BMP2- and, less potently, BMP4-induced suppression of androgen secretion but did not affect responses to BMP6 and BMP7. Noggin antagonized the effects of three BMPs (rank order: BMP4 > BMP2 > BMP7) but did not affect the response to BMP6. Follistatin partially reversed the suppressive effects of BMP6 on androgen secretion but did not affect BMP2, BMP4 and BMP7 action. Chordin had no effect on the response to any of the four BMPs. BMP6 treatment upregulated thecal expression of GREM1, NOG, CHRD and SMAD6 mRNA whilst inhibiting expression of the four BMPs. Taken together with previous work documenting the intra-ovarian expression of different BMPs, BMP binding proteins and signalling receptors, these observations reinforce the conclusion that extracellular binding proteins selectively modulate BMP-dependent alterations in thecal steroidogenesis. As such they likely constitute an important regulatory component of this, and other intra-ovarian actions of BMPs

The global effect of follicle-stimulating hormone and tumour necrosis factor α on gene expression in cultured bovine ovarian granulosa cells

Background Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone; FSH) and inhibitory (tumour necrosis factor alpha; TNFα) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3–6. Initially dose–response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNFα (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNFα, or with FSH plus TNFα (n = 4 per group) and RNA was harvested for microarray analyses. Results Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNFα/TNFα plus FSH. The effect of TNFα on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNFα being similar to those of FSH plus TNFα treatment. TNFα treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST, INHA, INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNFα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons γ and β1 and interleukin 1β. Conclusions In vitro, the transcriptome of granulosa cells responded minimally to FSH compared with the response to TNFα. The response to TNFα indicated an active process akin to tissue remodelling as would occur upon atresia. Additionally there was reduction in endocrine function and induction of an inflammatory response to TNFα that displays features similar to immune cells

Comparison of bioactivities, binding properties and intra-follicular levels of bovine follistatins

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, 41kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin- and heparan sulphate proteoglycan (HSP)-binding properties and bio-potencies in neutralization of activin-A action in vitro revealed that all five isoforms bound activin-A, but with different affinities. Only the 31kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency with 35 and 41kDa isoforms being least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analyzed bFF from dominant (DF) and subordinate (SF) follicles collected at strategic times during a synchronized estrous cycle. Total FST, activin-A and activin-AB were measured by immunoassay while individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with estrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin-A (r=-0.34), activin-AB (r=-0.80) and ‘total’ FST (r=-0.70) levels. Follicle diameter was positively correlated with abundance of the 41 kDa isoform (r=0.59) but negatively correlated with abundance of 33 and 31 kDa isoforms (r=-0.56, -0.41). Both follicle status (DF vs SF) and cycle stage affected total FST, activin-A, activin-B levels while follicle status, but not cycle stage, affected abundance of 41, 37, 33 and 31kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms that differ in their ability to bind and neutralise activins and associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may have an important negative role, either directly or via inhibition of the positive effects of activins, on follicle growth and function during follicular waves

The anti-epileptic drug Valproic Acid (VPA) inhibits steroidogenesis in bovine theca and granulosa cells in vitro

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC

Myostatin is expressed in bovine ovarian follicles and modulates granulosal and thecal steroidogenesis

Myostatin plays a negative role in skeletal muscle growth regulation but its potential role in the ovary has received little attention. Here, we first examined relative expression of myostatin (MSTN), myostatin receptors (ACVR1B, ACVR2B and TGFBR1) and binding protein, follistatin (FST), in granulosa (GC) and theca (TC) cells of developing bovine follicles. Secondly, using primary GC and TC cultures, we investigated whether myostatin affects steroidogenesis and cell number. Thirdly, effects of gonadotropins and other factors on MSTN expression in GC and TC were examined. MSTN, ACVR1B, TGFBR1, ACVR2B and FST mRNA was detected in both GC and TC at all follicle stages. Immunohistochemistry confirmed follicular expression of myostatin protein. Interestingly, MSTN mRNA expression was lowest in GC of large estrogen-active follicles while GC FST expression was maximal at this stage. In GC, myostatin increased basal CYP19A1 expression and estradiol secretion whilst decreasing basal and FSH-induced HSD3B1 expression and progesterone secretion and increasing cell number. Myostatin also reduced IGF-induced progesterone secretion. FSH and dihydrotestosterone had no effect on granulosal MSTN expression whilst insulin-like growth factor and tumour necrosis factor-alpha suppressed MSTN level. In TC, myostatin suppressed basal and LH-stimulated androgen secretion in a follistatin-reversible manner and increased cell number, without affecting progesterone secretion. LH reduced thecal MSTN expression whilst BMP6 had no effect. Collectively, results indicate that, in addition to being potentially responsive to muscle-derived myostatin from the circulation, myostatin may have an intra-ovarian autocrine/paracrine role to modulate thecal and granulosal steroidogenesis and cell proliferation/survival

Myostatin is expressed in bovine ovarian follicles and modulates granulosal and thecal steroidogenesis

Myostatin plays a negative role in skeletal muscle growth regulation but its potential role in the ovary has received little attention. Here, we first examined relative expression of myostatin (MSTN), myostatin receptors (ACVR1B, ACVR2B and TGFBR1) and binding protein, follistatin (FST), in granulosa (GC) and theca (TC) cells of developing bovine follicles. Secondly, using primary GC and TC cultures, we investigated whether myostatin affects steroidogenesis and cell number. Thirdly, effects of gonadotropins and other factors on MSTN expression in GC and TC were examined. MSTN, ACVR1B, TGFBR1, ACVR2B and FST mRNA was detected in both GC and TC at all follicle stages. Immunohistochemistry confirmed follicular expression of myostatin protein. Interestingly, MSTN mRNA expression was lowest in GC of large estrogen-active follicles while GC FST expression was maximal at this stage. In GC, myostatin increased basal CYP19A1 expression and estradiol secretion whilst decreasing basal and FSH-induced HSD3B1 expression and progesterone secretion and increasing cell number. Myostatin also reduced IGF-induced progesterone secretion. FSH and dihydrotestosterone had no effect on granulosal MSTN expression whilst insulin-like growth factor and tumour necrosis factor-alpha suppressed MSTN level. In TC, myostatin suppressed basal and LH-stimulated androgen secretion in a follistatin-reversible manner and increased cell number, without affecting progesterone secretion. LH reduced thecal MSTN expression whilst BMP6 had no effect. Collectively, results indicate that, in addition to being potentially responsive to muscle-derived myostatin from the circulation, myostatin may have an intra-ovarian autocrine/paracrine role to modulate thecal and granulosal steroidogenesis and cell proliferation/survival

A functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA and secreted protein level (75 and 94%, respectively) and elicited a 77% reduction in CYP17A1 mRNA level and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 mRNA level (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ~2-fold. The CYP17 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling

High-Precision Branching Ratio Measurement for the Superallowed + Emitter 74Rb

A high-precision branching-ratio measurement for the superallowed β + decay of 74Rb was performed at the TRIUMF Isotope Separator and Accelerator (ISAC) radioactive ion-beam facility. The scintillating electronpositron tagging array (SCEPTAR), composed of 10 thin plastic scintillators, was used to detect the emitted β particles; the 8π spectrometer, an array of 20 Compton-suppressed HPGe detectors, was used for detecting γ rays that were emitted following Gamow-Teller and nonanalog Fermi β + decays of 74Rb; and the Pentagonal Array of Conversion Electron Spectrometers (PACES), an array of 5 Si(Li) detectors, was employed for measuring β-delayed conversion electrons. Twenty-three excited states were identified in 74Kr following 8.241(4) × 108 detected 74Rb β decays. A total of 58 γ -ray and electron transitions were placed in the decay scheme, allowing the superallowed branching ratio to be determined as B0 = 99.545(31)%. Combined with previous half-life and Q-value measurements, the superallowed branching ratio measured in this work leads to a superallowed f t value of 3082.8(65) s. Comparisons between this superallowed f t value and the world-average-corrected Ft value, as well as the nonanalog Fermi branching ratios determined in this work, provide guidance for theoretical models of the isospin-symmetry-breaking corrections in this mass region.IS

High-precision branching-ratio measurement for the superallowed β\u3csup\u3e+\u3c/sup\u3e emitter 74Rb

A high-precision branching-ratio measurement for the superallowed β+ decay of 74Rb was performed at the TRIUMF Isotope Separator and Accelerator (ISAC) radioactive ion-beam facility. The scintillating electron-positron tagging array (SCEPTAR), composed of 10 thin plastic scintillators, was used to detect the emitted β particles; the 8π spectrometer, an array of 20 Compton-suppressed HPGe detectors, was used for detecting γ rays that were emitted following Gamow-Teller and nonanalog Fermi β+ decays of 74Rb; and the Pentagonal Array of Conversion Electron Spectrometers (PACES), an array of 5 Si(Li) detectors, was employed for measuring β-delayed conversion electrons. Twenty-three excited states were identified in 74Kr following 8.241(4)×108 detected 74Rb β decays. A total of 58 γ-ray and electron transitions were placed in the decay scheme, allowing the superallowed branching ratio to be determined as B 0=99.545(31)%. Combined with previous half-life and Q-value measurements, the superallowed branching ratio measured in this work leads to a superallowed ft value of 3082.8(65) s. Comparisons between this superallowed ft value and the world-average-corrected Ft̄ value, as well as the nonanalog Fermi branching ratios determined in this work, provide guidance for theoretical models of the isospin-symmetry-breaking corrections in this mass region. © 2013 American Physical Society