Fertilization and Early Embryogenesis
(W312) EGG ENVELOPE GLYCOPROTEIN ZP1 INDUCES SPERM ACROSOME REACTION IN JAPANESE QUAIL (Coturnix japonica).
Sasanami, Tomohiro1, Koyanagi, Kouji, Ohtsuki, Mamoru1, Kansaku, Norio3, Hiyama, Gen, Mori, Makoto1, 1 Shizuoka University, Shizuoka, Japan3 Azabu University, Sagamihara, Japan
ABSTRACT- The extracellular matrix surrounding avian oocytes, termed perivitelline membrane (PL), consists of at least two major glycoproteins. Our previous studies of Japanese quail have demonstrated that one of its components, ZPC, is synthesized in the ovarian granulosa cell. Another component, ZP1, is synthesized in the liver, and might be transported to the surface of the oocyte in the follicles. However, little is known about the role of these glycoproteins in fertilization. In order to understand the process of fertilization in bird, we evaluated the effects of ZP1 and ZPC on the induction of sperm acrosome reaction in Japanese quail. Ejaculated semen was obtained from male quail during natural mating with female, and was suspended in Hanks balanced salt solution containing 0.1% cloacal grand secretion. SDS-solubilized PL was separated on SDS-PAGE and the 175-kDa (dimeric ZP1), the 97-kDa (monomeric ZP1) and the 35-kDa (ZPC) bands were excised. The individual proteins were eluted by incubating the gel slices with 0.1% SDS buffered at pH 8.0 with 100 mM Tris-HCl overnight at 37°C. The eluant was then extensively dialyzed against water, lyophilized, and dissolved in 20 mM Tris-HCl (pH 8.0). Spermatozoa (final concentration, 1 x 107 spermatozoa /ml) were incubated at 39°C with or without the purified glycoprotein. After the incubation, sperm nucleus was stained with propidium iodide. Acrosome status was observed under the fluorescence microscopy based on the presence (acrosome-intact sperm) or absence (acrosome-reacted sperm) of the acrosome. When the ejaculated sperm were incubated with a calcium ionophore A23187, we found that the number of sperm without acrosome significantly increase in a dose-dependent manner. Treatment of spermatozoa with increasing dose of the purified dimeric ZP1 led to a concentration-dependent stimulation of acrosome reaction. The monomeric ZP1 had similar effect. Moreover, we found that the ZP1-induced acrosome reaction was significantly blocked by the pretreatment of the purified ZP1 with N-glycanase. On the other hand, the addition of the purified ZPC failed to induce acrosome reaction at any doses tested. Collectively, these results indicated that N-linked glycans on ZP1 play an essential role in triggering the acrosome reaction in Japanese quail.
KEY WORDS: Egg envelope, Acrosome reaction, ZP1, Japanese quail