|Ph.D Student||Shaked Orit|
|Subject||The Role of Human Sperm Metalloproteinases and Their|
Inhibitors in the Fertilization Process
|Department||Department of Medicine||Supervisor||Professor Zaki Kraiem|
The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade protein components of the extracellular matrix. These proteins, together with their tissue inhibitors (TIMPs), are involved in physiological processes, such as ovulation and embryo implantation in the uterus. Our purpose was to examine the identity and appearance and secretion profile of these enzymes as well as their contribution to the human fertilization process.
We have examined MMP and TIMP secretion in normal and OTA sperm using gel zymography and Western Blot analysis. Forty normal sperm samples and 40 abnormal sperm samples from men with oligo-terato-astheno-spermia syndrome (i.e. low sperm count, low motility and high percent of abnormal forms) were collected .Gel zymography showed 92-, 72-, 62-, and 28-kD molecular weight bands exhibiting gelating degrading activity in both normal and OTA sperm samples. A higher 28-kD activity and a lower 92kD MMP activity in normal sperm samples relative to OTA samples were detected. No marked difference in TIMP-1, -2, 72-kD, and 62-kD release was observed between normal and OTA sperm samples. The 92-, 72-, and 62-kD bands with gelatinolytic activity are consistent with pro-MMP-9, pro-MMP-2, and active MMP-2, respectively. A 28kD protein was identified as Acrosin which is known as a serine protease located in the acrosome membrane and secreted during the acrosome reaction (AR).
Another aspect of this work focused on the three signal transduction pathways: Protein Kinase A (PKA), Protein Kinase C (PKC) and Protein Tyrosine Kinase (PTK) and their involvement and effect on the different proteins. H-89, a PKA pathway inhibitor, lowered MMP-9 levels. Genistein, a PTK inhibitor, inhibited MMP-2 levels in normal sperm. Acrosin was not affected at all by the inhibitors. PKC pathway did not affect any MMP. Using different pathway inducers revealed that MMP-2 and pro-MMP-2 were not affected at all, while forskolin, which stimulates the PKA pathway, increased MMP-9 and acrosin levels. TPA, which stimulates the PTK pathway, increased MMP-9 levels in normal sperm.
MMPs and acrosin were then measured during the three stages in the fertilization process (capacitation, AR and fusion). MMP-2,-9 and acrosin were found present during the capacitation process. In addition, in OTA sperm MMP-9 levels were 4 times higher and Acrosin levels were 6 times lower when compared to normal sperm.
We found MMP-2,-9 and acrosin to be present in the sperm secretion during the AR. However, using gelatin activity assay, which measures specific protein activity, without separating it from the other sperm proteins, by degrading biotinylated gelatin, as opposed to zymography which measures the protein activity alone, by separating it from the other sperm proteins, revealed that the main active protein is acrosin. We also found that MMP-2,-9 are not involved in pro-acrosin activation which occurs at the spermatogenesis stage. Gelatin degrading activity in OTA sperm was significantly lower when compared to normal sperm.
MMP-2 and acrosin localization by immunoflurascence revealed their presence in the equatorial region, which fuses with the plasma membrane in the fusion stage. Experiments examining sperm ability to penetrate the oocyte in the presence or absence of specific inhibitors revealed that MMP-2,-9 and acrosin significantly inhibit sperm ability to penetrate the oocyte plasma membrane. OTA sperm was, as expected, not able to penetrate the oocyte plasma membrane.
In conclusion, this is the first report of MMP and TIMP secretion by normal and OTA human sperm. The results indicate a different profile between normal and OTA human sperm samples with higher acrosin and lower MMP-9 levels in normal versus OTA sperm. In addition, the model that we suggest indicates that MMP-2, -9 and acrosin are activated at different times during the fertilization process. While Acrosin is involved during all stages (capacitation, AR and fusion), MMP-2 and MMP-9 are only involved during the capacitation and fusion.