PARENT SESSION
SLIDE SESSION 16: FERTILIZATION
Chairs: William Breed, Nuch Tanphaichitr, Becky Sortini (Trainee)
Univ Ottawa-UCU Auditorium
1:30 PM-3:30 PM

FLUORESCENTLY-LABELED MURINE ZP3 AND ZP2 GLYCOPROTEINS BIND TO DISTINCT SITES ON SPERM IN A CALCIUM-, CAPACITATION-, AND MATURATION -DEPENDENT MANNER.

Kerr, Candace¹, Hanna, William¹, Shaper, Joel², Wright, William¹, ^{1 2}

ABSTRACT- Mammalian fertilization begins by the binding of sperm to a glycoprotein matrix surrounding the egg, called the zona pellucida (ZP). Characterization of zona proteins of the mouse egg has implicated ZP3 and ZP2 in the initial and secondary binding events between sperm and ZP, respectively. Despite the biological importance of these binding events, there has been limited characterization of ZP3 and ZP2 binding to murine sperm. For this purpose, we quantified the binding of isolated, solubilized ZP glycoproteins to murine sperm using quantitative fluorescent microscopy. Results showed that fluorescently-labeled ZP3 (Alexa-ZP3) bound over the acrosome of acrosome-intact (AI) sperm whereas fluorescently-labeled ZP2 (Alexa-ZP2) bound over the acrosomal region of acrosome-reacted (AR) sperm. However, Alexa-ZP3 and Alexa-ZP2 bound to the postacrosomal region of both AI and AR sperm heads, while binding of Alexa-ZP1 was not detected. Competitive binding assays comparing Alexa-ZP3 and Alexa-ZP2 binding in the presence of 10-fold molar excess of unlabeled ZP1, ZP2, ZP3 or solubilized total ZP demonstrated distinct sites for Alexa-ZP3 and Alexa-ZP2 on the sperm surface. Dose response analysis produced linear Scatchard plots of Alexa-ZP3 and Alexa-ZP2 binding with an estimated ~185,000 and 500,000 binding sites on sperm exhibiting ~67 nM and ~200 nM disassociation constants (K_d), respectively. Besides localizing and quantifying Alexa-ZP3 and Alexa-ZP2 binding on sperm, possible requirements for sperm-ZP binding were also studied including epididymal maturation, capacitation and Ca⁺². Results showed that Alexa-ZP3 did not bind caput sperm under capacitating conditions. Capacitation was a requirement for Alexa-ZP3 and total Alexa-ZP glycoproteins to bind sperm since neither bound caudae epididymal sperm under noncapacitating conditions. Additionally, Alexa-ZP3 and Alexa-ZP2 binding to capacitated caudae sperm decreased 80% when EGTA was added in 2.5-fold molar excess of the Ca⁺² present in the incubation media. In summary, this study identified discrete binding sites on the sperm surface for ZP3 and ZP2, which were dependent on Ca⁺², capacitation, and epididymal maturation of the sperm.

KEY WORDS: mouse, sperm, zona pellucida, QFM