265 DIFFERENTIATING X- AND Y-BEARING SPERMATOZOA ASSOCIATED WITH THE ZONA PELLUCIDA AT THE TIME OF FERTILIZATION

Abstract

In this laboratory, we are studying why a nutritionally complete maternal diet of high energy content can skew the sex ratio toward males in mice, and why a diet low in energy favors female offspring. One possibility is that the oocyte is able to attract or be fertilized by one class, i.e., X- or Y-bearing sperm relative to the other as a result of maternal diet during oogenesis. Alternatively, maternal diet may result in a reproductive tract environment that provides a competitive advantage for one class of sperm over the other. The objective of current experiment was to establish a fluorescent in situ hybridization (FISH) method for determining relative zona pellucida (ZP)-associated X and Y sperm number in control mice. While our laboratory and many others have employed FISH-based approaches to distinguish epididymal X and Y sperm, such procedures cannot be applied directly to ZP-associated sperm. Herein, we describe a successful method that maintains the integrity of this association and differentiates the ZP-associated X- versus Y-bearing sperm by FISH. Cumulus-oocyte complexes (COCs) were collected from NIH Swiss females on a control diet. Spermatozoa were obtained from the caudal epididymis of stud male mice, capacitated, and used to inseminate COCs under standard culture conditions. After 5 h, sperm/eggs were washed with PBS, transferred to a positively charged glass slide, and treated with 0.25 µL of 0.01 m HCl/0.1% Tween 20 solution at room temperature. The remaining structures, including attached spermatozoa, were fixed in 80% methanol (20 min, –20°C), subjected to microwave heating for 105 s, and treated with 0.01% pepsin for 20 min at 37°C, washed in 1 × SSC, fixed in 80% methanol, and air-dried. Hybridization probes (concentrated FITC-labeled X, Cy3-labeled Y; CamBio, Cambridge, UK) were combined and held at 65°C for 10 min and 37°C for 35 min before application to the slides at 37°C. Slides were sealed with coverslips and rubber cement, incubated at 75°C for 3 min, transferred to a pre-warmed humidified chamber, and incubated for 20 h at 37°C. Specimens were washed successively for 8 min twice in each of stringent conditions (50% formamide + 50% 1 × SSC, 45°C), 1 × SSC, and detergent solution, and then mounted and examined under a fluorescence microscope. A total of 20 oocytes with 1539 zona-associated sperm were analyzed. The average numbers of X and Y sperm per oocyte were 40.65 ± 4.23 and 36.30 ± 3.78, respectively, values not statistically different from 1:1. This FISH procedure appears to provide a valid and effective way to assess ratios of X andY sperm associated with the ZP. This procedure will be applied in future studies to determine whether ZP-associated X- versus Y-sperm number varies according to maternal diet in mice.

This work was supported by NIH Grant HD 044042.