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Vertebrate reproductive science and technology
RESEARCH ARTICLE

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

J. Mao, A. M. Davis, E. D. Fountain, R. M. Roberts and C. S. Rosenfeld

Reproduction, Fertility and Development 20(1) 212 - 213
Published: 12 December 2007

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.

https://doi.org/10.1071/RDv20n1Ab265

© CSIRO 2007

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