Growth factor receptor-bound protein 14 undergoes light-dependent intracellular translocation in rod photoreceptors: functional role in retinal insulin receptor activation.

Growth factor receptor-bound protein 14 (Grb14) is involved in growth factor receptor tyrosine kinase signaling. Here we report that light causes a major redistribution of Grb14 among the individual subcellular compartments of the retinal rod photoreceptor. Grb14 is localized predominantly to the inner segment, nuclear layer, and synapse in dark-adapted rods, whereas in the light-adapted rods, Grb14 redistributed throughout the entire cell, including the outer segment. The translocation of Grb14 requires photoactivation of rhodopsin, but not signaling through the phototransduction cascade, and is not based on direct Grb14-rhodopsin interactions. We previously hypothesized that Grb14 protects light-dependent insulin receptor (IR) activation in rod photoreceptors against dephosphorylation by protein tyrosine phosphatase 1B. Consistent with this hypothesis, we failed to observe light-dependent IR activation in Grb14(-/-) mouse retinas. Our studies suggest that Grb14 translocates to photoreceptor outer segments after photobleaching of rhodopsin and protects IR phosphorylation in rod photoreceptor cells. These results demonstrate that Grb14 can undergo subcellular redistribution upon illumination and suggest that rhodopsin photoexcitation may trigger signaling events alternative to the classical transducin activation.

Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle.

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.

Phosphoinositide 3-kinase signaling in retinal rod photoreceptors.

PURPOSE: Phosphoinositide 3-kinase (PI3K) consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The biological significance of PI3K was investigated in vertebrate rod photoreceptors by deleting its regulatory p85α protein and examining its role in photoreceptor structure, function, and protein trafficking. METHODS: Mice that expressed Cre recombinase in rods were bred to mice with a floxed p85α (pik3r1) regulatory subunit of PI3K to generate a conditional deletion of pik3r1 in rods. Functional and structural changes were determined by ERG and morphometric analysis, respectively. PI3K activity was measured in retinal homogenates immunoprecipitated with an anti-PY antibody. Akt activation was determined by Western blot analysis with a pAkt antibody. RESULTS: Light-induced stress increased PI3K activity in retinal immunoprecipitates and phosphorylation of Akt. There was no effect of pik3r1 deletion on retinal structure. However, twin flash electroretinography revealed a slight delay in recovery kinetics in pik3r1 knockout (KO) mice compared with wild-type controls. The movement of arrestin in the pik3r1 KO mice was slower than that in the wild-type mouse retinas at 5 minutes of exposure to light. At 10 minutes of exposure, the ROS localization of arrestin was almost identical between the wild-type and pik3r1 KO mice. CONCLUSIONS: The results provide the first direct evidence that rods use PI3K-generated phosphoinositides for photoreceptor function. The lack of phenotype in pik3r1 KO rod photoreceptors suggests a redundant role in controlling PIP(3) synthesis.

Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: influence of follicle size on responses to GDF9.

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.

Effects of feeding two levels of propionibacteria to dairy cows on plasma hormones and metabolites.

To determine the effect of feeding propionibacteria on metabolic indicators during lactation, multiparous and primiparous Holstein cows were fed one of three dietary treatments in a 2 x 3 factorial design from 2 weeks prepartum to 30 weeks post partum: (1) Control (primiparous n=5, multiparous n=8) fed a total mixed ration (TMR); (2) high-dose group (primiparous n=6, multiparous n=5) fed TMR plus 6 x 10 (11) cfu/head daily (high-dose P169) of propionibacterium strain P169; or (3) low-dose group (primiparous n=8, multiparous n=6) fed TMR plus 6 x 10(10) cfu/head daily (low-dose P169) of P169. Blood samples were collected weekly and analysed for plasma concentrations of glucose, insulin, insulin-like growth factor-I (IGF-I), leptin, nonesterified fatty acids (NEFA) and cholesterol. Between weeks 25 and 30, all groups received bovine somatotropin (bST) every 2 weeks. Low-dose P169 multiparous cows had lower (P<0.05) plasma insulin and glucose concentrations than high-dose P169 multiparous cows, whereas high-dose P169 primiparous cows had lower glucose but greater insulin concentartions than low-dose P169 primiparous cows (P<0.05). Plasma insulinratioglucose molar ratios were 13-18% lower (P<0.05) in low-dose P169 cows than in control or high-dose P169 cows. Plasma IGF-I, NEFA and leptin levels did not differ among diet groups between weeks 1 and 25. Low-dose P169 multiparous cows had 25% greater plasma cholesterol levels than high-dose P169 and control multiparous cows, but cholesterol levels in primiparous cows did not differ. During bST treatment, high-dose P169 multiparous cows and low-dose P169 primiparous cows had lower IGF-I levels than their respective controls and, regardless of parity, high-dose P169 cows had greater NEFA than control cows. Although supplemental feeding of P169 altered plasma hormones and metabolites, the particular effects were dependent on dose of P169 and parity of cows.