Oocyte-mediated suppression of follicle-stimulating hormone- and insulin-like growth factor-induced secretion of steroids and inhibin-related proteins by bovine granulosa cells in vitro: Possible role of transforming growth factor alpha

The objective was to investigate the potential role of the oocyte in modulating proliferation and basal, FSH-induced and insulin-like growth factor (IGF)-induced secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E-2), and progesterone (P-4) by mural bovine granulosa cells. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin and androstenedione, and the effects of ovine FSH and IGF analogue (LR3-IGF-1) were tested alone and in the presence of denuded bovine oocytes (2, 8, or 20 per well). Medium was changed every 48 h, cultures were terminated after 144 h, and viable cell number was determined. Results are based on combined data from four independent cultures and are presented for the last time period only when responses were maximal. Both FSH and IGF increased (P < 0.001) secretion of inh A, act A, FS, E-2, and P-4 and raised cell number. In the absence of FSH or IGF, coculture with oocytes had no effect on any of the measured hormones, although cell number was increased up to 1.8-fold (P < 0.0001). Addition of oocytes to FSH-stimulated cells dose-dependently suppressed (P < 0.0001) inh A (6-fold maximum suppression), act A (5.5-fold), FS (3.6-fold), E-2 (4.6-fold), and P-4 (2.4-fold), with suppression increasing with FSH dose. Likewise, oocytes suppressed (P < 0.001) IGF-induced secretion of inh A, act A, FS, and E-2 (P < 0.05) but enhanced IGF-induced P-4 secretion (1.7-fold; P < 0.05). Given the similarity of these oocyte-mediated actions to those we observed previously following epidermal growth factor (EGF) treatment, we used immunocytochemistry to determine whether bovine oocytes express EGF or transforming growth factor (TGF) alpha. Intense staining with TGFalpha antibody (but not with EGF antibody) was detected in oocytes both before and after coculture. Experiments involving addition of TGFalpha to granulosa cells confirmed that the peptide mimicked the effects of oocytes on cell proliferation and on FSH- and IGF-induced hormone secretion. These experiments indicate that bovine oocytes secrete a factor(s) capable of modulating granulosa cell proliferation and responsiveness to FSH and IGF in terms of steroidogenesis and production of inhibin-related peptides, bovine oocytes express TGFalpha but not EGF, and TGFalpha is a prime candidate for mediating the actions of oocytes on bovine granulosa cells.

Differential changes in transforming growth factor β isoform expression during postnatal cardiac growth

Transforming growth factor-β (TGF-β) is synthesised as an inactive precursor protein; this is cleaved to produce the mature peptide and a latency associated protein (LAP), which remains associated with the mature peptide until activation by LAP degradation. Isoform specific antibodies raised against the LAPs for TGF-β2and -β3were used to determine the myocardial levels of LAP (activatable TGF-β) and full length precursor (inactive TGF-β) forms during post-natal development in the rat. TGF-β2was present predominantly as the precursor in 2 day old myocardium. There was an age-dependent shift from precursor protein to LAP between 2 and 28 days. A corresponding increase in the level of mature (activatable) TGF-β2was found. TGF-β3was detected in significant quantities only as LAP. However, a four-fold increase in the expression of TGF-β3LAP was observed between 2 and 28 days. The substantial increases in activatable forms of TGF-β2and -β3that occur in myocardium during the first 28 days of life in the rat support a role for these proteins in post-natal cardiac development.

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, RMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. in addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.(C) 2003 Elsevier Science B.V. All rights reserved.

Inhibition of 125I-epidermal growth factor binding to murine fibroblasts and keratinocytes by irradiation with ultraviolet-B light

Treatment of murine Swiss 3T3 fibroblasts and XB/2 keratinocytes with UV-B light (302 nm) resulted in a dose-dependent inhibition of [125I] epidermal growth factor (EGF) binding. The light dose required to achieve 50% inhibition of binding in both cell types was 80–85 J/m2 Decreased [125I] platelet-derived growth factor binding was not evoked even by light doses of up to 280 J/m2 UV-B irradiation did not stimultate phosphorylation of the 80 kd protein substrate for protein kinase C. Furthermore, its effect on [125I]EGF binding was not altered as a consequence of protein kinase C down-regulation following prolonged exposure of cells to phorbol esters. These results indicate that UV-B-induced transmodulation of the epidermal growth factor receptor is a specific event mediated through a protein kinase C-indepen dent pathway. Transfer of culture medium from irradiated cells to untreated control cells showed this effect was not induced as a result of transforming growth factor α release and subsequent binding to the EGF receptor in these cells.

Morphology and myofiber composition of skeletal musculature of the forelimb in young and aged wild type and myostatin null mice

Most current research into therapeutic approaches to muscle diseases involves the use of the mouse as an experimental model. Furthermore, a major strategy to alleviate myopathic symptoms through enhancing muscle growth and regeneration is to inhibit the action of myostatin (Mstn), a transforming growth factor-beta (TGF-beta) family member that inhibits muscle growth. Presently, however, no study has expanded the morphological analysis of mouse skeletal muscle beyond a few individual muscles of the distal hindlimb, through which broad conclusions have been based. Therefore, we have initially undertaken an expansive analysis of the skeletal musculature of the mouse forelimb and highlighted the species-specific differences between equivalent muscles of the rat, another prominently used experimental model. Subsequently, we examined the musculature of the forelimb in both young and old adult wild-type (mstn(+/+)) and myostatin null (mstn(-/-)) mice and assessed the potential beneficial and detrimental effects of myostatin deletion on muscle morphology and composition during the aging process. We showed that: (1) the forelimb muscles of the mouse display a more glycolytic phenotype than those of the rat; (2) in the absence of myostatin, the induced myofiber hyperplasia, hypertrophy, and glycolytic conversion all occur in a muscle-specific manner; and, importantly, (3) the loss of myostatin significantly alters the dynamics of postnatal muscle growth and impairs age-related oxidative myofiber conversion.

Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles

Ovarian follicle development is regulated through endocrine and local mechanisms. Increasing evidence indicates roles for transforming growth factor beta superfamily members, including inhibins and activins. We recently identified divergent expression of mRNAs encoding activin receptors (ActR) and inhibin co-receptor betaglycan in chicken follicles at different stages of maturation. Here, we compare the actions of LH and FSH (0, 1, 10, 100 ng/ml) on levels of mRNA for ActRI, ActRIIA, ActRIIB and betaglycan in chicken granulosa and theca cells (GC and TC) from preovulatory (F1) and prehierarchical (6-8 mm) follicles. The expression of mRNAs for LH-R and FSH-R and production of inhibin A, oestradiol and progesterone were also quantified. FSH decreased ActRIIB and ActRI mRNA levels in 6-8 mm GC, whereas LH increased the mRNA levels. Both LH and FSH enhanced ActRIIA (5- and 8.5-fold) and betaglycan mRNA expression (2- and 3.5-fold) in 6-8 mm GC. In 6-8 mm TC, LH and FSH both increased the betaglycan mRNA level (7- and 3.5-fold respectively) but did not affect ActRI, ActRIIA and ActRIIB transcript levels. In F1 GC, both LH and FSH stimulated ActRI (2- and 2.4-fold), ActRIIB (3.2- and 2.7-fold) and betaglycan (7- and 4-fold) mRNA levels, while ActRIIA mRNA was unaffected. In F1 TC, LH and FSH reduced ActRIIA (35-50%) and increased (4.5- and 7.6-fold) betaglycan mRNA, but had no effect on ActRI and ActRIIB transcript levels. Results support the hypothesis that expression of ActR and betaglycan are differentially regulated by gonadotrophins during follicle maturation in the hen. This may represent an important mechanism for fine-tuning follicle responsiveness to local and systemic activins and inhibins.

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

Objectives: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. Methods: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We. determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. Results: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. Conclusions: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

S6K1 is acetylated at lysine 516 in response to growth factor stimulation

The 70kDa ribosomal protein S6 kinase 1 (S6K1) plays important roles in the regulation of protein synthesis, cell growth and metabolism. S6K1 is activated by the phosphorylation of multiple serine and threonine residues in response to stimulation by a variety of growth factors and cytokines. In addition to phosphorylation, we have recently shown that S6K1 is also targeted by lysine acetylation. Here, using tandem mass spectrometry we have mapped acetylation of S6K1 to lysine 516, a site close to the C-terminus of the kinase that is highly conserved amongst vertebrate S6K1 orthologues. Using acetyl-specific K516 antibodies, we show that acetylation of endogenous S6K1 at this site is potently induced upon growth factor stimulation. Although S6K1 acetylation and phosphorylation are both induced by growth factor stimulation, these events appear to be functionally independent. Indeed, experiments using inhibitors of S6K1 activation and exposure of cells to various stresses indicate that S6K1 acetylation can occur in the absence of phosphorylation and vice versa. We propose that K516 acetylation may serve to modulate important kinase-independent functions of S6K1 in response to growth factor signalling.

Relationships between the plasma concentrations of insulin-like growth factor-I in dairy cows and their fertility and milk yield

The relationships between insulin-like growth factor-I (IGF-I) and the fertility and milk yield of Holstein-Friesian dairy cows were investigated. The concentration of IGF-I in blood was measured weekly from one week before to 12 weeks after calving in 177 multiparous cows and at four times during this period in 142 primiparous cows; the concentration of IGF-I in milk was measured in 50 of the multiparous cows. The plasma concentrations of IGF-I were higher in the primiparous than in the multiparous animals. in the primiparous cows, high concentrations of IGF-I before calving were associated with longer calving to conception intervals. Conversely, in the multiparous cows low concentrations of IGF-I before and after calving were associated with a failure to conceive, despite repeated services. Multiparous cows with IGF-I concentrations of greater than 25 ng/ml in the week after calving were 11 times more likely to conceive to first service than those with lower concentrations. Concentrations of IGF-I greater than 50 ng/ml at first service increased the likelihood of conception five-fold. Cows with higher peak milk yields had lower plasma concentrations of IGF-I and took longer to return to ovarian cyclicity. The negative relationship between milk yield and return to cyclicity was stronger in the multiparous cows (P<0(.)002) than in the primiparous cows (P<0(.)04). The concentrations of IGF-I in milk followed a different pattern and were not associated with the changes in plasma IGF-I or fertility.