Loss of Fyn kinase Impairs Oocyte Quality and Developmental Potential.

Abstract

Fyn kinase is highly expressed in mammalian and non-mammalian oocytes and has been implicated in the signal transduction events including calcium signaling, cell cycle events such as meiosis resumption and pronuclear congression. The results of the above experimental studies contradict other reports that the Fyn-null mice exhibit normal fertility. The purpose of the present study was to compare the fertility of B6/129S7-Fyntm1Sor/J female mice with females from the parent strain (B6/129SF2/J) (The Jackson Laboratory, Bar Harbor, ME). Western blot analysis revealed the absence of Fyn protein in Fyn-null oocytes, although these oocytes did over express Yes protein kinase, possibly in an attempt to compensate for the loss of Fyn. Fyn-null females were smaller in size than controls and produced significantly smaller litter sizes during the first five parturitions. The interval between litters was longer and the overall reproductive lifespan was shorter than controls. Superovulation of age-matched females produced oocytes that were similar in number and size to controls, but exhibited a thinner zona pellucida and a marked accumulation of dense cytoplasmic inclusions of unknown composition. Evaluation by confocal microscopy revealed that approximately 18% of the ovulated eggs recovered from the oviduct at 15hrs post hCG administration were immature (MI) which was significantly higher than controls. Fyn-null oocytes also exhibited defects in spindle organization as well as reduced ability to position the MII spindle close to the egg cortex. These spindle abnormalities correlated with defects in cortical actin cytoskeleton polarity. Fertilization in vitro with wild type sperm revealed that the Fyn-null oocytes were fertilized at a rate similar to controls, but exhibited reduced developmental potential since 73% of fertilized eggs (pronuclei) reached the 2-cell stage vs 98% from controls. Embryos that reached the 2-cell stage developed to blastocyst at a rate similar to controls. Calcium imaging studies revealed that Fyn-null oocytes fertilized with wild type sperm exhibited reduced frequency of calcium oscillations and a modified pattern of peak amplitude, although the total calcium signal produced by Fyn-null oocytes was similar to controls. The altered calcium signaling properties were correlated with a modified distribution of IP3 receptor as detected by confocal immunofluorescence. The IP3 receptor type 1 was frequently distributed in a highly irregular pattern in Fyn-null oocytes with large clumps or accumulations that were not present in control oocytes. In summary, the Fyn-null oocyte exhibits defects in meiotic maturation, meiotic spindle integrity, cortical cytoskeleton organization, and calcium signaling that may explain the reduced developmental potential. (platform)