PARENT SESSION
FERTILIZATION

Monday, August 2, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(239) SPERM BINDING TO UTERINE AND OVIDUCTAL EPITHELIAL EXPLANTS, ZONA PELLUCIDA, AND FERTILIZATION CAPACITY IN VITRO.

Siddiqui, Mohammed^{1, 2}, Brenny, Suzanne¹, Kaya, Abdullah^{1, 3}, Enwall, Lefric¹, Shamsuddin, Mohammed², Parrish, John¹, ¹ University of Wisconsin, Madison, WI² Bangladesh Agricultural University, Mymensingh, Bangladesh³ University of Selçuk, Konya, Kampüs, Turkey

ABSTRACT- We hypothesized the sperm binding to uterine epithelial cells (UEC), oviductal epithelial cells (OEC) or zona pellucida (ZP) would be related to bull in vitro fertility status. Bull fertility and sperm binding tests were conducted on sperm from 5 different bulls exposed to UEC-, OEC explants (2x2 mm2), ZP, and used for in vitro fertilization (IVF). DNA fluorochrome (Hoechst 33342) and digital image analysis technique were employed to measure sperm binding per unit surface area of UEC, OEC and ZP. At 1, 3, 6, 9 and 22h sperm-tissue incubation terminated, tissue explants fixed and squashed individually onto a micro chambered (100 or 800 M depth) slide. Tissue-bound sperm were counted by fluorescent illumination (excitation: 365 nm, emission: 460 nm, barrier filter: 400). In vitro fertilization was performed and development of embryos at 135 h was evaluated by fluorescent marker. A batch of oocytes were incubated with 5-fold diluted sperm of same preparation used in IVF and incubation terminated at 2 h. Sperm bound oocytes were fixed, squashed, and fluorescent images of the top surface of the oocytes were acquired by a photometric SenSys camera. The image of each of the oocytes was processed, surface area measured and the number of sperm counted by using ImageJ (a public domain image analysis software by NIH). The mean number of sperm bound per sq. mm surface of epithelium of the body, horn, isthmus and ampulla of the female tract were 4, 5, 39 and 23, respectively. Sperm binding is significantly higher (P<0.01) in isthmus than the other parts of the female reproductive tract at individual bull cases. However, there was no bull difference in respect to sperm binding to the uterine or oviductal explants (p>0.05). The fertilization rate and embryo development rates of the fertilized embryos were 60, 55, 55, 54 and 42, and 63, 61, 51, 42 and 48, respectively for each bull. The sperm binding per 10x103 M2 of surface area of ZP were 14, 13, 12, 11 and 9, respectively for each bull. One of the 5 bulls differs significantly (P<0.05) with the others with respect to zona binding assay (ZBA) or fertilization rate. There was a significant (P<0.05) linear relationship in that ZBA increases with increasing IVF. This digital image analysis technique needs to be evaluated for its ability to predict in vivo bull fertility.

KEY WORDS: FERTILIZATION, EXPLANTS, SPERM, OVIDUCT