Oocytic expression of zona pellucida protein ZP 4 in Japanese quail ( Coturnix

1 The avian perivitelline layer, an extracellular matrix homologous to the zona pellucida 2 of mammalian oocytes, is composed mainly by zona pellucida gene family glycoproteins. 3 Our previous studies in Japanese quail have demonstrated that the matrix's components, 4 ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is 5 synthesized in the liver. Recently, we demonstrated that another minor constituent, ZP2 is 6 produced in the oocytes of the immature follicles. In the present study, we report the 7 isolation of cDNA encoding quail ZP4 and its expression and origin in the female birds. 8 By RNase protection assay and in situ hybridization, we demonstrated that ZP4 transcripts 9 were transcribed in the oocytes of small white follicles. The expression level of ZP4 10 decreased dramatically during follicular development, and the highest expression was 11 observed in the small white follicles. Western blot analysis using the specific antibody 12 against ZP4 indicated that the immunoreactive 58.2 kDa protein was present in the lysates 13 of the small white follicles. These results demonstrate for the first time that the avian ZP4 14 is expressed in the oocyte, and that the expression pattern of the gene is similar to that of 15 ZP2. 16 17 Keywords: egg envelope, Japanese quail, zona pellucida, ZP4,. 18 19 3 INTORODUCTION 1 The egg envelope is an extracellular matrix that surrounds the oocyte of vertebrate 2 animals and plays indispensable roles in gamete recognition during the initial process of 3 fertilization, including species-specific sperm-egg binding, induction of the acrosome 4 reaction and prevention of polyspermy (McLeskey et al. 1998). These egg envelopes are 5 mainly constructed of glycoproteins belonging to different subclasses of the zona pellucida 6 (ZP) gene family that possess a highly conserved amino acid sequence near the C terminus 7 (Hoodbhoy et al. 2005), human (Lefievre et al. 2004), bonnet monkey (Ganguly et al. 11 2008) and hamster (Izquierdo-Rico et al. 2009) zona pellucida, 4 glycoproteins have been 12 identified. However, in the case of mouse zona pellucida, only 3 glycoproteins have been 13 identified because mouse ZP4 is a pseudogene due to the accumulation of stop codons 14 (Conner et al. 2005). Amino acid sequence analysis reveals that ZP4 is most closely 15 related to ZP1 because there is a trefoil domain in the upstream of the ZP domain (Conner 16 et al. 2005). This indicate that …