The Action of JAK/STAT3 and BMP/HJV/SMAD Signaling Pathways on Hepcidin Suppression by Tucum-do-Cerrado in a Normal and Iron-Enriched Diets.

The Brazilian savanna fruit, tucum-do-cerrado (Bactris setosa Mart.) reduces hepatic hepcidin levels. Therefore, we investigated the effect of tucum-do-cerrado on the TfR/HFE and/or BMP/HJV/SMAD and JAK/STAT pathways, in normal and excess iron conditions. Rats were treated with: control diet (CT); control diet +15% tucum-do-cerrado (Tuc); iron-enriched diet (+Fe); or iron-enriched diet +15% tucum-do-cerrado (Tuc+Fe). Tucum-do-cerrado (Tuc) decreased hepatic Hamp and Hjv mRNA levels but did not alter Bmp6, Smad7, Tfr1, and Hfe mRNA levels; pSMAD1/5/8 and pSTAT3 protein levels; labile iron pool (LIP); and inflammatory biomarkers, compared to the CT group. The iron-enriched diet increased Hamp mRNA levels, as well as pSMAD1/5/8 and pSTAT3 protein levels, while no difference was observed in Hjv, Bmp6, Smad7, Tfr1, and Hfe mRNA levels and LIP compared to the CT group. The association of tucum-do-cerrado with the iron-enriched diet (Tuc+Fe) decreased Hamp, Hjv, Bmp6, and Hfe mRNA levels and pSTAT3 protein content compared to the +Fe group, while increased Hamp and decreased Hfe mRNA levels compared to the Tuc group. Therefore, the inhibition of hepatic hepcidin by tucum-do-cerrado consumption may involve the downregulation of intestinal Dmt1 and hepatic Hjv expression and deacetylation mediated by SIRT1 by a mechanism that is independent of tissue iron content. However, in excess iron conditions, the modulation of hepatic hepcidin expression by tucum-do-cerrado seems to be partially mediated by the inflammatory signaling pathway, as well as involves the chelating activity of tucum-do-cerrado.

BMP2, 4 and 6 and BMPR1B are altered from early stages of bovine cystic ovarian disease development.

Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1.

BMP6 and BMP4 expression in patients with cancer-related anemia and its relationship with hepcidin and s-HJV.

In the present study, we investigated BMP6 and BMP4 expression in patients with cancer-related anemia (CRA) as well as its relationship with hepcidin and s-HJV. The avidin-biotin system enzyme-linked immunosorbent assay was used to test serum levels of BMP6, BMP4, s-HJV, and hepcidin in 53 cancer patients with anemia and 52 control cancer patients without anemia. Serum levels of BMP6 and hepcidin in the anemia group were 434.53 ± 212.11 ng/mL and 5.68 ± 3.89 µg/L, respectively. In the non-anemia cancer group, serum BMP6 and hepcidin levels were 334.37 ± 171.32 ng/mL and 4.60 ± 2.28 µg/L, which were significantly lower than the levels for the CRA group (P < 0.05). In addition, the serum level of s-HJV was 0.69 ± 0.28 ng/mL in the CRA group, which was significantly lower compared to that for the non-anemia group (1.07 ± 1.00 ng/mL, P < 0.01). There were no significant differences in BMP4 expression between the two groups. BMP6 was negatively correlated with s-HJV and Hb (r = -0.2536 and -0.2949, P < 0.01), but was not correlated with hepcidin. Similarly, BMP4 expression was not correlated with Hb, s-HJV, or hepcidin. Our study shows that patients with CRA had high expression of BMP6 and hepcidin and low expression of s-HJV. BMP6 was found to be negatively correlated with s-HJV; both regulate hepcidin expression and play important roles in the development of anemia.

Levels of BMP-6 mRNA in goat ovarian follicles and in vitro effects of BMP-6 on secondary follicle development.

Expression of BMP-6 mRNA was quantified by real-time polymerase chain reaction (PCR) and the BMP-6 protein was demonstrated by immunohistochemistry in the primordial, primary, secondary, small and large antral follicles of goat. Furthermore, the influence of BMP-6 on increase in diameter, antrum formation and expression of BMP-6 and FSH-R in in vitro cultured secondary follicles was studied. Therefore, goat primordial, primary and secondary follicles, as well as small and large antral follicles were obtained and the mRNA levels of BMP-6 were quantified by PCR in real time. Expression of BMP-6 protein in goat follicles was demonstrated by immunohistochemistry. The influence of BMP-6 in the presence or absence of follicle-stimulating hormone (FSH) on both the development of secondary follicles and the expression of mRNA for BMP-6 and FSH-R was evaluated after 6 days of culture. Furthermore, the follicular diameter and the formation of the antrum were evaluated before and after 6 days of culture and compared by Kruskal-Wallis and chi-squared tests (P < 0.05), respectively. The results show that the level of mRNA for BMP-6 in primary and secondary follicles was significantly higher than in the primordial follicles (P < 0.05). Similar levels of BMP-6 mRNA were observed in cumulus-oocyte complexes and mural granulosa/theca cells from small and large antral follicles, respectively. BMP-6 protein was expressed in oocytes of all categories of follicles and in granulosa cells from secondary follicles onwards. Addition of BMP-6 to the culture medium increased the diameter of secondary follicles mainly by antrum formation after 6 days' culture, in the presence or absence of FSH (P < 0.05). Furthermore, addition of FSH resulted in increased levels of BMP-6 mRNA in these follicles (P < 0.05). Simultaneous administration of FSH and BMP-6 enhanced the levels of FSH receptor (FSH-R) mRNA (P < 0.05). It is concluded that BMP-6 mRNA is increased during transition from primordial to primary/secondary follicles in the goat ovaries and that BMP-6 enhances the growth of cultured secondary follicles.

Stability of housekeeping genes and expression of locally produced growth factors and hormone receptors in goat preantral follicles.

The aim of the present study was to investigate the stability of six housekeeping genes, and the relative expression of growth factors (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 and KL) and hormone receptors (FSH, LH and GH) in goat preantral follicles. To evaluate to stability of housekeeping genes micro-dissected fresh follicles (150-200 µm) as well as follicles that have been in vitro cultured for 12 days were used. In addition, isolated fresh follicles were used to compare expression of various growth factors and hormone receptors before culture. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA. After amplification of cDNA by real-time PCR, the geNorm software program was used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), ß-tubulin, ß-actin, phosphoglycerokinase (PGK), 18S rRNA, ubiquitin (UBQ) and ribosomal protein 19 (RPL-19). In addition, follicular steady-state levels of mRNA from the various growth factors under study were compared. Results demonstrated that, in goat preantral follicles, UBQ and ß-actin were the most suitable reference genes and thus could be used as parameters to normalize data from future in vitro studies. In contrast, 18S RNA appeared the least stable gene among the tested housekeeping genes. Analysis of mRNA for several hypophyseal hormone receptors in fresh preantral follicles showed significantly higher FSH-R mRNA levels than those of LH-R and GH-R, and no difference between GH-R and LH-R mRNA levels. In regard growth factor mRNA expression in goat preantral follicles, EGF mRNA levels appeared significantly lower than those of the other studied growth factors. Increasingly higher relative mRNA levels were observed for GDF-9, BMP-15, BMP-6, FGF-2, VEGF, Kl and IGF-1, successively. In conclusion, UBQ and ß-actin are the most stable housekeeping genes in fresh and 12-days cultured caprine preantral follicles. Furthermore, in fresh follicles, high levels of FSH-R mRNA are detected while among eight growth factors, IGF-1 is the most highly expressed and EGF the weakest expressed compound.