H.B.A. Bastos

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.

Ultrastructural and histological characteristics of the endometrium during early embryo development in mares

The aim of this study was to evaluate ultrastructural and histological changes in the endometrium on days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. Mares were routinely examined by transrectal palpation and ultrasonographic examination of the reproductive tract until estrus was detected. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mares uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. From ovulation detection until day of uterine biopsy, blood samples to measure Progesterone concentrations were collected daily in cyclic and pregnant mares. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7-13. On the 7th day of pregnancy a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. The concentrations of P4 were affected by day (P < 0.001), but were not affected by group. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications may aid to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation.

Early Embryo Development in Mares: Proteomics of Uterine Fluid

The expression and regulation of endometrial proteins is crucial for conceptus implantation and development. However, little is known about site-specific proteomic profiles of the mammalian endometrium during the peri-implantation period [1]. The conceptus-derived signals used for pregnancy recognition have been identified in nearly all of the important domestic animal species; one notable exception is the horse [2]. Proteomic descriptions of the uterine fluid of early pregnant mares are scant. The aim of this study was to compare the proteomic profile of uterine fluid on days 7, 10 and 13 after ovulation in pregnant and cyclic mares.

Gene Expression of MMP-1, MMP-2 and TNF-α in the Endometrium of Mares With Different Degrees of Fibrosis

The endometrial health is a determinant factor for mare fertility. However, equine endometrosis is characterized by glandular alterations and periglandular fibrosis in the endometrial stroma leading to uterine physiology malfunction [1]. Tissue fibrogenesis occurs when there is a disaccord between degradation and synthesis of components of the extracellular matrix, mediated by specific proteinases [2]. In mammals the selective expression, activation or inhibition of specific groups of Matrix Metalloproteinases (MMPs) modulates the process of uterine remodeling during various phases of the estrous cycle, peripartum and postpartum periods [3]. The pathogenesis of uterine fibrosis has not yet been fully elucidated, but is related to the age of the mares [4]. Higher degrees of endometrosis cause changes in the pattern of endometrial cellular interaction that decreases the ability to carry the pregnancy to term [1]. Cell signaling is mediated by cytokines, such as TNF-a, which have the ability to regulate fibroblasts and to produce MMPs [5]. The study of MMPs and TNF-a may elucidate important points of the development of endometrial fibrosis; contributing to the classification methods of the fibrotic process and find a new marker of endometrial fibrosis. This study aimed to quantify the gene expression of matrix metalloproteinases 1 and 2 (MMP-1; MMP-2) and Tumor Necrosis Factor (TNF-a) in the endometrium of mares with different degrees of fibrosis.

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.

The Role of PLCζ and WPB2NL Gene Expression in Semen Quality and Fertility of Stallions

In the last decades, there was an increase in the investigation of equine reproduction to maximize both genetic gain and competition traits in the horse industry [1], however, little is known about the influence of genetic factors in equine fertility [2]. Oocyte activation at fertilization in all mammalian species studied is brought about by repetitive increases in intracytoplasmic calcium (Ca2+) concentration, referred to as Ca2+, oscillations, triggered by the testis-specific protein isoform phospholipase C zeta (PLCz), which is delivered by the sperm at the time of fertilization [3]. The oscillation of Ca2+ levels plays an essential role in early embryonic development [4], moreover, a decrease of PLCz was demonstrated in a subfertile stallion [5]. Recently, PAWP, sperm-born tryptophan domain-binding protein coded by WBP2NL has been proposed as an alternative or complementary pathway for oocyte activation based on findings that injection of recombinant PAWP induced Ca2+ oscillations comparable to those of fertilization [6]. However to our knowledge the expression of WBP2NL was not described in stallion spermartozoa. The aim of this study was to verify the influence of PLCz and WBP2NL gene expression in sperm cells in relation to semen quality and fertility in stallions.

Equine Sperm Selection by Synthetic Membrane Filter

&NA; To improve the quality of artificial insemination doses, the selection of spermatozoa that are most likely to achieve fertilization from the rest of the ejaculate is an alternative approach. The present study aimed to determine the kinetics and plasma membrane integrity and functionality after sperm selection with a synthetic membrane filter in polyvinyl chloride chambers with two different diameters and two filtrations times. Twelve ejaculates from three stallions were used. Immediately after collection, semen was diluted (T0) and analyzed. Two different chambers were made with two elbows connected with a pipe divided by a 5‐&mgr;m pore synthetic membrane filter. One chamber had an inner diameter of 26 mm and the other, 36 mm. Skim milk at 37°C was placed in a side of the chamber (A). In the other side of the chamber (B), a sample of the extended semen, with known number of spermatozoa, was deposited. After 7 and 15 minutes, a sample was obtained from the “A” side of each chamber, and sperm concentration was calculated, semen analyzed by computer‐assisted motility analysis, and plasma membrane functionality and integrity evaluated. Total motility, progressive motility, and plasma membrane integrity were improved (P < .05) after filtration in both devices and filtration times. Concentration was lower (P < .05) in both chambers at all times in relation to T0 semen. The filtration device demonstrates to be a practical and easy alternative for sperm selection. Selection using the chambers allows an increase in kinetics and membrane integrity and functionality independent of time and device diameter. HighlightsSperm selection using a 5‐&mgr;m pore synthetic membrane.Chamber diameter and filtration times evaluated.Both devices increased sperm quality.