基质金属蛋白酶-2,-9及其组织抑制因子在恒河猴黄体中的表达Matrix metalloproteinases-2,-9 and their tissue inhibitors expression in Macaca mulatta corpus luteumAbstract:Matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) are critical proteolytic enzymes involved in a variety of physiological and pathological processes in the ovary, including corpus luteum formation and regression. Tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) are also important regulators of MMP activity. The present study aimed to investigate the expression patterns of MMP-2, MMP-9 and their inhibitors in Macaca mulatta corpus luteum (CL) during different stages of the menstrual cycle. Immunohistochemistry and quantitative real-time PCR were employed to analyze the expression of these proteins in the CL of the ovary. We found that MMP-2 and MMP-9 were expressed at high levels in the developing and mid-cycle luteal phases while their inhibitors, TIMP-1 and TIMP-2, were up-regulated in the late luteal phase. Our results suggest that MMP-2 and MMP-9 play important roles in the maintenance and regression of M. mulatta corpus luteum, and inhibitors TIMP-1 and TIMP-2 may act as the key regulators of their activity.Key Words: Matrix metalloproteinase, tissue inhibitor of metalloproteinase, corpus luteum, Macaca mulattaIntroduction:The corpus luteum (CL) is a transient endocrine gland that forms after ovulation under the stimulation of luteinizing hormone (LH), and is responsible for the production of progesterone, which is essential for the maintenance of pregnancy in mammals (1). The formation and regression of CL are tightly controlled by a variety of factors, including cytokines, growth factors, and extracellular matrix (ECM) remodeling enzymes such as matrix metalloproteinases (MMPs) (2, 3). MMPs are a family of proteinases that can degrade ECM components, including collagens, laminins, and fibronectin, and play important roles in tissue remodeling, cancer invasion, and angiogenesis (4).MMPs are secreted as inactive proproteins that are activated by proteolytic cleavage of the propeptide domain. Among the MMP family members, MMP-2 and MMP-9, also known as gelatinases, are capable of degrading type IV collagen and gelatins, and are involved in the degradation of the basement membrane during angiogenesis and tissue remodeling (5). The activity of MMPs is tightly regulated by their endogenous inhibitors, known as tissue inhibitors of metalloproteinases (TIMPs), which bind to their active site and prevent their proteolytic activity (6). TIMPs are a family of four members, with TIMP-1 and TIMP-2 being the most prominent inhibitors of MMP-2 and MMP-9 (7).Although the roles of MMP-2, MMP-9, and their inhibitors TIMP-1 and TIMP-2 in the CL have been widely studied in other species, the expression patterns and their functional roles during different stages of the menstrual cycle remain unclear in Macaca mulatta. Therefore, the purpose of this study was to investigate the expression patterns of MMP-2, MMP-9 and their inhibitors in M. mulatta CL during different stages of the menstrual cycle.Materials and Methods:Animals:M. mulatta females were housed in the animal facility of Kunming Primate Research Center. The menstrual cycles of the animals were monitored by measuring serum progesterone levels, and CL tissues were collected from the ovaries at different stages of the menstrual cycle. This study was approved by the Animal Care and Use Committee of Kunming Primate Research Center.Immunohistochemistry:Frozen sections of M. mulatta CL were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and blocked in 5% bovine serum albumin for 1h. The sections were incubated with anti-MMP-2, anti-MMP-9, anti-TIMP-1 or anti-TIMP-2 antibodies (1:100 dilution; Abcam, USA) overnight at 4, followed by incubation with secondary antibodies (1:200 dilution; Jackson ImmunoResearch, USA) for 1h at room temperature. After washing with PBS, the sections were stained with DAPI and visualized using a fluorescence microscope.Quantitative real-time PCR:Total RNA was isolated from M. mulatta CL tissues using TRIzol (Invitrogen, USA) according to the manufacturers instructions. The RNA was reverse-transcribed to cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, USA) and quantitative real-time PCR was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, USA) with a Power SYBR Green PCR Master Mix (Applied Biosystems, USA). The primer sequences of MMP-2, MMP-9, TIMP-1, TIMP-2, and GAPDH are shown in Table 1. GAPDH was used as an internal control for normalization.Statistical analysis:Data were analyzed using GraphPad Prism software (version 7.0) and expressed as mean SEM. One-way ANOVA followed by Tukeys multiple comparison test or Students t-test was used for statistical analysis. P 0.05 was considered statistically significant.Results: