Gene expression of matrix metalloproteinases and LH receptors in mare follicular development.

The period from the emergence of a dominant follicle until its formation requires tissue remodeling. Enzymes promoting collagen lysis, such as matrix metalloproteinases (MMPs), are fundamental for the process of extracellular matrix remodeling, which allows changes in ovarian tissue architecture during follicular growth. It has been suggested that the production of these enzymes may be affected by the rise in circulating concentrations of LH, which acts on the ovarian surface epithelium (OSE). The aim of this study was to determine the expression of MMP-1, MMP-2, and LH receptor (LHR) in the ovulation fossa and in the central portion of the equine ovary during follicular deviation and dominance. Ovaries of 12 cyclic mares were selected and subsequently divided into two groups: development (DEV) group and dominant (DOM) group. The DEV group consisted of ovaries from six animals whose follicles were less than 28 mm in diameter (follicular deviation), and the DOM group consisted of ovaries from six animals whose follicles measured 28 mm or more in diameter (dominant follicles). The latter group was divided into two subgroups: the group of ovaries with a dominant follicle (DOM-D) and the group of contralateral ovaries (DOM-C). Our results showed that mRNA for MMP-1, MMP-2, and LHR was present in the equine ovary during follicle development, in the ovulation fossa, and in the central portion of the ovary. MMP-1 and LHR gene expression was greater (P < 0.05) for the DOM-D group compared with the DOM-C group. In the DOM-D group, MMP-1, MMP-2, and LHR gene expression was greater (P < 0.05) in the ovarian stroma compared with the ovulation fossa. Using immunohistochemistry, OSE from the DOM group showed increased expression compared with the DEV group (P < 0.05). In conclusion, we demonstrated that MMP-1 and MMP-2 might be fundamental for events related to tissue remodeling, which occurs during follicular development until the formation of the dominant follicle. We also demonstrated the relationship between the gene expression of MMPs and the gene and protein expression of LHR, suggesting that LHR in the OSE might be an important factor to initiate the signaling cascade that culminates with the production of MMPs.

Cytotoxic effect of amphotericin B in a myofibroblast cell line.

In this study we investigate whether Amphotericin B (AmB), a widely used antifungal agent, could decrease the proliferation of a myofibroblast cell line - GRX, a model of activated hepatic stellate cells (HSC). Three different hepatic cell lines (GRX, Hep G2 and ARL-6) were treated with two concentrations of AmB (1.25 µg/mL or 2.50 µg/mL). Cytotoxicity was assessed by MTT assay. The effects of AmB on GRX migration was evaluated by Wound-healing Assay. Cell cycle arrest was investigated by flow cytometry. Apoptosis and autophagy were analyzed by Caspase 3 and LC3 immunostaining, respectively. Treatment with AmB 1.25 or 2.50 µg/mL showed a decrease in viability of GRX cells. This decrease was not observed for Hep G2 or ARL-6 in any of the two AmB concentrations tested. GRX cells treated with 1.25 µg/mL AmB were unable to close the wound after 96 h. Cell cycle analysis showed an increase in sub-G1 population and a decrease in G2/M population in AmB-treated cells. In addition, AmB-treated GRX cells showed increased expression of LC-3 and Caspase-3 by immunohistochemistry, suggesting an increase in both autophagy and apoptosis. Here we show that AmB is cytotoxic for GRX cells, a model of activated HSC, but not for hepatic lineages HepG2 and ARL6.