Effect of bone morphogenetic proteins 2 and 4 on survival and development of bovine secondary follicles cultured in vitro.

This study evaluated the effect of bone morphogenetic proteins 2 (BMP2) and 4 (BMP2) on follicle development and mRNA expression for GDF9, Cyclin B1, BMPR1A, BMPR1B, BMPRII, FSHR and SMAD1 in bovine secondary follicles cultured in vitro. Isolated secondary follicles were cultured for 18 days in TCM199+ medium alone or supplemented with BMP2 (10 ng/mL), BMP4 (100 ng/mL) or combination of both BMP2 and 4. Real-time PCR was used to analyze mRNA levels in fresh and cultured follicles. After 18 days of culture, follicles cultured with BMP2 alone or with BMP4 alone had larger diameters when compared to control (P < .05). In addition, all treatments promoted antrum formation and maintained a high viability rate through the growing period. The presence of BMP2, BMP4 or both together did not influence mRNA expression for the tested genes. However, the in vitro culture induces down-regulation for mRNA expression of BMPR1A. In conclusion, the addition of BMP2 or BMP4 alone in cultured medium promotes follicular growth and antrum formation in bovine follicles after 18 days of in vitro culture.

Bovine ovarian stem cells differentiate into germ cells and oocyte-like structures after culture in vitro.

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte-like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.

Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1ß on primordial follicle activation and survival in vitro.

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1ß on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1ß (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1ß, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1ß (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1ß promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.

Alkaloids and volatile constituents from the stem of Fusaea longifolia (Aubl.) Saff. (Annonaceae)

A phytochemical study of the ethanol extract and an extraction of the volatile compounds, performed by means of Clevenger apparatus were carried out with the stem of Fusaea longifolia (Aubl.) Saff. (Annonaceae). The ethanol extract yielded O-methylmoschatoline, isolated for the first time in this species, and stepholidine, reported for the first time in genus Fusaea. The structural identification of the alkaloids was made based on the analysis of their NMR spectra. Through the use of GC and GC-MS, two sesquiterpenoids, a-cadinol (12.5 percent) and spatulenol (12.0 percent) were identified as the major constituents of the essential oil.

Outros constituintes químicos de Diplotropis ferruginea Benth. (Fabaceae)

O extrato hexânico das cascas do caule de Diplotropis ferruginea Benth (Fabaceae), após procedimentos cromatográficos e fitoquímicos, forneceu o triterpeno lupeol, citado pela primeira vez em Diplotropis ferruginea, e etil-2-hidroxi-4-methoxi-6-propil-benzoate, descrito pela primeira vez em na literatura. As determinações estruturais desses compostos foram realizadas com base em análises espectroscópicas de ¹H e 13C NMR, bem como também por comparação com valores da literatura.

The hexane extract of the stem bark of Diplotropis ferruginea Benth (Fabaceae), after chromatografic and phytochemical procedures, yielded the triterpene lupeol, wich has been cited for the first time on Diplotropis ferruginea, and ethyl-2-hydroxy-4-methoxy-6-propyl-benzoate, described for the first time on literature. The structural determinations of these compounds were performed on the basis of ¹H and 13C NMR spectroscopic analysis as well as comparison with literature values.

Two amides from Piper tuberculatum fruits.

The fruits of Piper tuberculatum yielded pellitorine, N-isobutyl-2E,4E-decadienamide (1), and piperidide-2E,4E-decadienamide (2). Their complete NMR analysis, based on one- and two-dimensional experiments, is reported.

Bowdenol, a new 2,3-dihydrobenzofuran constituent from Bowdichia virgilioides.

From the stem bark of Bowdichia virgilioides, lupeol, lupeol acetate, sitosterol, stigmasterol and methyl-3-[2-(1-hydroxymethylvinyl)-2,3-dihydrobenzo[b]furan-5-yl]-(E)-2-propenoate have been isolated. The latter is a new compound and it has been named as bowdenol. Their structures were elucidated with the aid of spectroscopic techniques, mostly 1 and 2D NMR. The biogenetic pathway for bowdenol has been suggested.

Krametosan, a new trinorlignan from the roots of Krameria tomentosa.

A new phenylcoumarone type trinorlignan, krametosan (1), along with the known norlignans, ratanhiaphenol I (2) and 2-(2'-hydroxy-4',6'-dimethoxyphenyl)-5-(E)-propenylbenzofuran (3), the lignan conocarpan (4) and dinorlignan decurrenal (5), were isolated from the CHCl3 extract of the roots of Krameria tomentosa. The structure of these compounds were elucidated by the spectroscopic methods.

Tetrahydroprotoberberine and aporphine alkaloids from rollinia leptopetala.

Tetrahydrojatrorrhizine, discretamine, anonaine and roemerine were isolated from the roots of Rollinia leptopetala R. E. Fries. They were identified and characterized by nuclear magnetic resonance spectroscopy ( 1 H and 13 C) with the aid of two-dimensional techniques ( 1 H- 1 H COSY, HETCOR, HMQC and HMBC). With the exception of tetrahydrojatrorrhizine, the presence of these alkaloids in R. leptopetala has not been reported previously.