Search for the genes involved in oocyte maturation and early embryo development in the hen

<p>Abstract</p> <p>Background</p> <p>The initial stages of development depend on mRNA and proteins accumulated in the oocyte, and during these stages, certain genes are essential for fertilization, first cleavage and embryonic genome activation. The aim of this study was first to search for avian oocyte-specific genes using an <it>in silico </it>and a microarray approaches, then to investigate the temporal and spatial dynamics of the expression of some of these genes during follicular maturation and early embryogenesis.</p> <p>Results</p> <p>The <it>in silico </it>approach allowed us to identify 18 chicken homologs of mouse potential oocyte genes found by digital differential display. Using the chicken Affymetrix microarray, we identified 461 genes overexpressed in granulosa cells (GCs) and 250 genes overexpressed in the germinal disc (GD) of the hen oocyte. Six genes were identified using both <it>in silico </it>and microarray approaches. Based on GO annotations, GC and GD genes were differentially involved in biological processes, reflecting different physiological destinations of these two cell layers. Finally we studied the spatial and temporal dynamics of the expression of 21 chicken genes. According to their expression patterns all these genes are involved in different stages of final follicular maturation and/or early embryogenesis in the chicken. Among them, 8 genes (<it>btg4</it>, <it>chkmos</it>, <it>wee</it>, <it>zpA</it>, <it>dazL</it>, <it>cvh</it>, <it>zar1 </it>and <it>ktfn) </it>were preferentially expressed in the maturing occyte and <it>cvh</it>, <it>zar1 </it>and <it>ktfn </it>were also highly expressed in the early embryo.</p> <p>Conclusion</p> <p>We showed that <it>in silico </it>and Affymetrix microarray approaches were relevant and complementary in order to find new avian genes potentially involved in oocyte maturation and/or early embryo development, and allowed the discovery of new potential chicken mature oocyte and chicken granulosa cell markers for future studies. Moreover, detailed study of the expression of some of these genes revealed promising candidates for maternal effect genes in the chicken. Finally, the finding concerning the different state of rRNA compared to that of mRNA during the postovulatory period shed light on some mechanisms through which oocyte to embryo transition occurs in the hen.</p

Elevated serum IGF-1 levels synergize PTH action on the skeleton only when the tissue IGF-1 axis is intact

Abstract There is growing evidence that IGF-1 and PTH have synergistic actions on bone and that part of the anabolic effects of PTH are mediated by local production of IGF-1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF-1. We utilized mice carrying a hepatic IGF-1 transgene (HIT), which results in a 3-fold increase in serum IGF-1 levels and normal tissue IGF-1 expression, and IGF-1 null mice with blunted IGF-1 expression in tissues but 3-fold increases in serum IGF-1 levels (KO-HIT). Evaluation of skeletal growth showed that elevations in serum IGF-1 in mice with igf-1 gene ablation in all tissues except the liver (KO-HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross-sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF-1 and PTH on bone. In sharp contrast, although PTH-treated KO-HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared to vehicle treated KO-HITs, their response was identical to that of PTH-treated control mice. We conclude that 1) in the presence of elevated serum IGF-1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF-1 and, 2) elevations in serum IGF-1 levels synergize PTH action on bone only if the tissue IGF-1 axis is intact, thus enhancement of PTH anabolic actions is tissue IGF-1-dependent

Serum IGF-1 Affects Skeletal Acquisition in a Temporal and Compartment-Specific Manner

Insulin-like growth factor-1 (IGF-1) plays a critical role in the development of the growing skeleton by establishing both longitudinal and transverse bone accrual. IGF-1 has also been implicated in the maintenance of bone mass during late adulthood and aging, as decreases in serum IGF-1 levels appear to correlate with decreases in bone mineral density (BMD). Although informative, mouse models to date have been unable to separate the temporal effects of IGF-1 depletion on skeletal development. To address this problem, we performed a skeletal characterization of the inducible LID mouse (iLID), in which serum IGF-1 levels are depleted at selected ages. We found that depletion of serum IGF-1 in male iLID mice prior to adulthood (4 weeks) decreased trabecular bone architecture and significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th) by 16 weeks (adulthood). Likewise, depletion of serum IGF-1 in iLID males at 8 weeks of age, resulted in significantly reduced transverse cortical bone properties (Ct.Ar, Ct.Th) by 32 weeks (late adulthood), but had no effect on trabecular bone architecture. In contrast, depletion of serum IGF-1 after peak bone acquisition (at 16 weeks) resulted in enhancement of trabecular bone architecture, but no significant changes in cortical bone properties by 32 weeks as compared to controls. These results indicate that while serum IGF-1 is essential for bone accrual during the postnatal growth phase, depletion of IGF-1 after peak bone acquisition (16 weeks) is compartment-specific and does not have a detrimental effect on cortical bone mass in the older adult mouse

MALDI Mass Spectrometry Imaging of Lipids and Gene Expression Reveals Differences in Fatty Acid Metabolism between Follicular Compartments in Porcine Ovaries

In mammals, oocytes develop inside the ovarian follicles; this process is strongly supported by the surrounding follicular environment consisting of cumulus, granulosa and theca cells, and follicular fluid. In the antral follicle, the final stages of oogenesis require large amounts of energy that is produced by follicular cells from substrates including glucose, amino acids and fatty acids (FAs). Since lipid metabolism plays an important role in acquiring oocyte developmental competence, the aim of this study was to investigate site-specificity of lipid metabolism in ovaries by comparing lipid profiles and expression of FA metabolism-related genes in different ovarian compartments. Using MALDI Mass Spectrometry Imaging, images of porcine ovary sections were reconstructed from lipid ion signals for the first time. Cluster analysis of ion spectra revealed differences in spatial distribution of lipid species among ovarian compartments, notably between the follicles and interstitial tissue. Inside the follicles analysis differentiated follicular fluid, granulosa, theca and the oocyte-cumulus complex. Moreover, by transcript quantification using real time PCR, we showed that expression of five key genes in FA metabolism significantly varied between somatic follicular cells (theca, granulosa and cumulus) and the oocyte. In conclusion, lipid metabolism differs between ovarian and follicular compartments

Additional file 8: of Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Figure S7. Signaling pathways in bovine granulosa cells after DHA or TUG-891 treatment. Effects of DHA or TUG-891 on phosphorylation of (A) mitogen-activated protein kinase 14 (MAPK14), (B) AMP-activated protein kinaseα (AMPKα) and (C) protein kinase B (Akt) signaling pathways were assessed in bovine granulosa cells cultured for 15 h in enriched McCoy’s 5A media with 10 μM DHA or 1 μM TUG-891, as described in Material and Method section for 5, 10, 30 and 60 min. Protein extracts were separated by electrophoresis on 4–12% (w:v) SDS-polyacrylamide gel. After electrotransfer to nitrocellulose membranes, the proteins were probed with anti-phosphorylated (p-) MAPK14 (A), anti-p-AMPKα (B) or anti-p-AKT1/2/3 (C) antibodies. The blots were stripped and re-probed with antibodies against MAPK14, AMPKα or Akt, respectively. The blots presented are representative of the quantification reported in Fig. 8. (TIF 228 kb

Additional file 4: of Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Figure S3. Gene expression of (A) free fatty acid receptor 1 (FFAR1) and (B) free fatty acid receptor 4 (FFAR4) in bovine ovarian cells. Total mRNA was extracted from the ovarian cortex (CX), thecal cells (TH), granulosa cells (GC) and cumulus cells (CC). Total mRNA was then reverse-transcribed and real-time RT-PCR was performed. The geometric mean of two housekeeping genes (RPL19- ribosomal protein L19 and RPS9- ribosomal protein S9) was used to normalize gene expression. Results of 2 to 4 independent samples are presented as means ± SEM. Bars with different superscripts are significantly different (p < 0.05). (TIF 59 kb

Additional file 1: of Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Figure S1. Control of customized free fatty acid receptor 4 (FFAR4) antibody specificity. Protein extracts from bovine lung tissue were separated by electrophoresis on 4–12% (w:v) SDS-polyacrylamide gel. After electrotransfer to nitrocellulose membranes, the proteins were probed with anti-FFAR4 antibody (0.95 μg/mL, customized FFAR4 rabbit antibody, Agro-Bio), which was pre-incubated for 15 min with different concentrations of the bovine specific peptide (Agro-Bio) used to produce the antibody (from 0 to 2.5 μg/mL). The blots were stripped and re-probed with antibodies against vinculin (VCL) used as the loading control. (TIF 93 kb

Additional file 2: of Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Table S1. Characteristics of primary antibodies used for western blotting and / or immunohistochemistry or immunofluorescence. (DOCX 16 kb

Additional file 7: of Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Figure S5. Signaling pathways in bovine granulosa cells after treatment with other concentrations of DHA (50 μM) or TUG-891 (10 and 50 μM). Effects of DHA or TUG-891 on phosphorylation of (A) mitogen-activated protein kinase 14 (MAPK14), (B) AMP-activated protein kinaseα (AMPKα) and (C) mitogen-activated protein kinase 1/3 (MAPK1/3) signaling pathways were assessed in bovine granulosa cells cultured for 15 h in enriched McCoy’s 5A media with 50 μM DHA or with 10 or 50 μM TUG-891, as described in Material and Method section for 5, 10, 30 and 60 min. Protein extracts were separated by electrophoresis on 4–12% (w:v) SDS-polyacrylamide gel. After electrotransfer to nitrocellulose membranes, the proteins were probed with anti-phosphorylated (p-)MAPK14 (A), anti-p-AMPKα (B) or anti-p-MAPK1/3 (C) antibodies. The blots were stripped and re-probed with antibodies against MAPK14, AMPKα, or MAPK1/3, respectively. Bands on the blots were quantified. Results of four independent experiments are presented as the ratio of p-protein to total protein, normalized by the ratio observed in control at each time and expressed as mean ± SEM, with time 0 min being equal to 1 (for reference). Bars with different superscripts are significantly different (p < 0.05). (TIF 99 kb