Functional Study Of Protein Alcyltransferase ZDHHC3 During Oocyte Maturation

Gametogenesis is required for sexual reproduction in higher organisms, such as vetebrates. Meiosis is an essential process to produce gametes for fertilization and to maintain genome stability in many species. Fully grown oocytes of most vertebrates are arrested at the prophase I of meiosis (also called G2 arrest). G2 arrest is a key checkpoint for oocyte meiosis. Upon exposed to stimuli, such as progesterrone, oocytes resume meiotic process, also called G2/M transition. The regulation of G2/M transition is essential for oocyte quality and fertilization later on. Underlying mechanisms of the G2 arrest and release remain to be fully elucidated.G2/M transition is known to be regulated by the G protein couple receptor signaling pathway (GPCR-Gas-Adenylyl cyclase-cAMP). Previously, we showed that long chain fatty acyl-CoA synthetase acs11b is required for maintaining the meiotic arrest in Xenopus. Acsllb presumably provides palmitoyl-CoA which can be utilized by downstream acyltransferases to modify proteins essential for the G2 arrest. In the present study, I report that protein acyltransferase ZDHHC3 functions downstream of acsllb to maintain oocyte meiotic arrest. Depletion of maternal ZDHHC3 RNA in fully-grown oocytes causes G2/M transition at very low concentration (0.01μM) progesterone which is insufficient to induce the transition in untreated oocytes. As expected, Gas palmitoylation level is greatly decreased in ZDHHC3 depleted oocytes. Furthermore we mapped ZDHHC3 palmitoylation sites in Gas and showed that palmitoylation deficient Gas failed to arrest oocytes at G2. We also identified a critical residue in ZDHHC3 critically required for its palmitoylation activity towards Gas. Thus, we conclude that ZDHHC3 is a key enzyme to palmitoylate proteins to maintain G2 arrest in Xenopus oocytes.To further study protein palmitoylation in mouse oocyte, I firstly used protein palmitoylation inhibitors to treat denuded mouse oocytes in the presence of 2.5μM milrinone, which inhibits hydrolysis of cAMP in oocyte. Inhibition of general protein palmitoylation caused meiotic resumption in mouse oocyte, while adenylyl cyclase activator forskolin could partially block the effect of inhibitor. Loss of function of acyltransferases ZDHHC3/7 also caused meiotic resumption of mouse oocyte. Furthermore, inhibition of protein palmitoylation affected the plasma membrane targeting of Gas targeting to plasma membrane. These data suggest that ZDHHC3/7 may also be involved in meiotic arrest through regulating Gas palmitoylation in mouse oocyte. To further study the physiological function of ZDHHC3/7, I constructed ZDHHC3/7 knockout mouse. However, there was no effect on female fertility in ZDHHC3 or ZDHHC7 single knockout mice, but ZDHHC3/7 double knockout mouse cannot survive. The results suggested that ZDHHC3 and ZDHHC7 are redundant in early mouse embryonic development. Functions of ZDHHC3 and ZDHHC7 for mouse oocyte arrest require future investigations through oocyte specific gene deletion.Taken together, protein acyltransferase ZDHHC3 plays an essential role in maintain GPCR signaling, thus meiotic arrest of Xenopus oocytes. Further studies are warranted to elucidate whether protein palmitoylation is reversibly regulated in GPCR signaling during oocyte G2/M transition. Our findings will contribute to better understanding the process of oocyte development and are important for elucidating the regulation of protein palmitoylation.