MMP-2, MMP-9, MT1-MMP, TIMP-1 和 TIMP-2基質金屬蛋白酶於犬造精過程表現及其精液品質關係之研究

Abstract

Semen is routinely collected from male dogs for the purposes of artificial insemination. Seminal plasma (SP) of mammal is a physiological secretion from multiple glands of the male reproductive tract that plays an important role in the final maturation of the spermatozoa through hormonal, enzymatic and surface-modifying events, and it functions as a vehicle for ejaculated spermatozoa. This complex mixture differs among species and contains a variety of biochemical components. Previous studies have identified matrix metalloproteinases (MMPs) as one of the proteins expressed in human, pig, ram and horse seminal plasma. However, the identity and function of MMPs in canine semen remains unknown. The functions of MMPs in spermatogenesis cannot be overlooked. The dissertation was aimed to investigate the role of matrix metalloproteinase (MMP-2, MMP-9) in determining semen quality and to evaluate the expression and cellular localization of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n=21) and abnormal (n=25) semen samples showed a significant, six-fold increase in proMMP-2 and MMP-2 activity in high than low sperm concentration samples (P<0.001). ProMMP-9 and MMP-9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (P<0.001). High levels of proMMP-2 and MMP-2 were associated with high sperm motility (≥ 70%, P < 0.001). Sperm rich fraction showed significantly (eight-fold) higher proMMP-9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 were confirmed in testicular and epididymal tissues. Testes from healthy mongrel dogs (N = 12; age 3.92±1.73 year olds) were fixed in Modified Davidson’s solution. Immunohistochemistry was performed and ten fields per specimen were evaluated in semi-quantitative scores. The stained slides were scored representing estimated area in proportion (P; 0 to 5 scales) and intensity of positive-staining cells (I; 0 to 3 scales). Total score (TS) was the sum of P and I. Sertoli cell expressed MMP-2, MMP-9, MT1-MMP and TIMP-2. Spermatogonia showed the strongest intensive staining for MT1-MMP (TS=8). Staining for MT1-MMP (TS=0) was absent in spermatocyte, spermatid and elongated spermatids. The strongest cytoplasm immunostaining of pachytene spermatocytes (TS=8), round and elongating spermatids (TS=8) were observed for MMP-9. TIMP-2 showed compact positive in the cytoplasmic granule of pachytene spermatocyte in all stages (TS=7). More importantly, MMP2 was mainly localized (TS=8) in the perinuclear region of round spermatids, representing the position of the developing acrosome. Moreover, the acrosome region of elongated spermatids detected only MMP2 protein (TS=8). Immunolabeling of epididymal and ejaculated sperm demonstrated MMP-2 localization along acrosomal region of sperm while MMP-9, TIMP-1, and TIMP-2 localization was merely limited to the flagella. This is the first report demonstrating the specific localization of MMP-2, MMP-9, MT1-MMP and TIMP-2 in canine spermatogenesis. In summary, the data indicate a high MMP-2 profile in normal sperm sample and a high MMP-9 profile in abnormal sperm samples. The enzymatic activity of MMP-2 and MMP-9 may serve as an alternative biomarker in determining semen quality. The spermatozoa initially acquire MMPs and TIMPs during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. MMP-2 might represent a new member of enzymes, which function in vesicle trafficking from the Golgi apparatus to the acrosome during spermatogenesis. The necessity of breakdown of physical barriers in the fertilization process suggests that MMP-2 might be concerned in this task.