G.O. Santos

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.

Combination of hCG and Deslorelin Acetate on the Induction of Ovulation in Mares: Changes in Follicular Fluid Protein Profile

The composition of follicular fluid (FF) is essential for the proliferation and differentiation of granulosa cells in addition to processes related to rupture of the follicular wall, maturation and fertilization of the oocyte and luteinization. In the mare, there are few studies evaluating FF proteome (FAHIMINIYA, Prot Sci, 9:1, 2011; PETRUCCI, J Eq Vet Sci, 34:115, 2014). The ability to induce ovulation in a reliable way is important in equine reproductive management in different situations the main ovulation-inducing agents used are hCG and deslorelin. The aim of this study was to compare the protein profile of FF on induced ovulation of mares with hCG or with the combination of hCG and deslorelin acetate. Fourteen mares were used (3-12 years). Following the observation of follicles ≥ 35 mm and with endometrial edema, the mare was submitted to the induction protocols: Group H 1000 UI, IV, of hCG or Group HG 1000 UI hCG, IV, + 1,5 mg of Deslorelin acetate, IM. In the subsequent cycle mares were submitted to a protocol different from the previous cycle. Samples were collected by transvaginal aspiration 32 h after induction and submitted to the quantification of proteins by the Bradford method. Two-dimensional electrophoresis was performed in 12.5% polyacrylamide gel and stained with Coomassie G250, scanned and analyzed using PDQuest v.8.0.1. Spots with significant differences in relative abundance between group H and HG were cut out submitted to trypsin digestion and mass spectrometry. In this study the total protein concentration in the mare FF from Group HG were higher (73.07 ± 6.42 mg/ml) than those induced with hCG alone (63.97 ± 6.97 mg/ml). Comparative analysis showed a significant difference in the abundance of five spots between groups. Two Alpha-1-antiproteinase 2 (A1AT2), the Serotransferrin (TF) and Antithrombin III (ATIII) had lower relative expression in group HG and the Haptoglobin (HP) showed greater abundance in the same group. The lowest expression of A1AT2 at the final moment of follicular maturation prior to ovulation is likely related to the need for lower inhibitory action on the proteolytic activity allowing fine adjustment that controls the ECM degradation, inflammation and the coagulation cascade (BIANCH, J Prot, 90:61, 2013). ATIII is also serine-type endopeptidase inhibitor activity. The last protein less expressed in the group HG was TF. Increase in cellular iron levels stimulates the expression of some MMPs that degrade the ECM. There are reports of increased transferrin in granulosa cells and oocyte follicles in more advanced stages of maturation, which could explain the reduction in FF transferrin. Haptoglobulin showed increased abundance in the group HG and exerts anti-inflammatory action due to inhibition of oxidative damage. These proteins are probably related to the final events of oocyte/follicle maturation that trigger ovulation and subsequent luteinization.

Do Therapeutic Doses of Phenilbutazone Affect Ovulation Processes in Mares

Ovulation is the unique process by which mature (preovulatory) ovarian follicles respond to the surge of luteinizing hormone (LH) and rupture to release fertilizable oocytes [1]. LH initiates and synchronizes a series of biochemical events that culminate in the breakdown of the follicle wall and extrusion of the oocyte. However, ovulation is associated with an inflammatory type reaction with an increase in the synthesis of inflammatory cytokines, prostaglandins and cortisol in the ovulatory follicle, and the rupture of the follicle wall that requires the presence of proteolytic enzymes degrading the extracellular matrix [2]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are probably the most frequently used analgesic agents in horses worldwide, primarily because many of the commonly occurring causes of pain are mediated by inflammation [3]. The primary mechanism of action of NSAIDs is inhibition of cyclooxygenase enzymes which are involved in the production of prostaglandins [4]. Phenylbutazone (PB), flunixin-meglumine (FM) and ketoprofen remain the most commonly used NSAIDs in equine medicine [4]. Nevertheless, one study showed that systemic intravenous administration of high doses of FM to mares during the periovulatory period blocked ovulation and induced luteinized unruptured follicles (LUF) in 83% of treated mares [5]. The aim of this study was to determine if different treatments with therapeutic doses of PB affect ovulation processes in mares.