Usage of X- and Y-chromosome fluorescent in situ hybridization to determine whether the murine oocytes selectively attract one class of spermatozoa over another.

While gender in mammals is determined by heterogametic male germ cells, controversy exists whether female reproductive tract selectively permits one class of sperm to reach the oocyte. Additionally, the oocyte itself might be able to attract or selectively bind to X- or Y-bearing sperm, but disagreement exists on this subject. In a recent study, investigators employed PCR analysis with sex chromosome specific primers and demonstrated that bovine oocytes were fertilized with equal frequency by X- or Y-bearing sperm (Zuccotti et al. 2005). However, this study permitted each oocyte to be fertilized by more than one spermatozoa. Tthese results might not mirror normal physiological conditions. Another study demonstrated no skewing of sex ratio in bovine oocytes in vitro fertilized with sorted or mixed sperm populations (Bermejo-Alvarez et al. 2008). To address this question in rodents, we sought to establish a fluorescent in situ hybridization (FISH) method for determining relative oocyte-associated X and Y sperm numbers in control mice. While our laboratory and many others have employed FISH-based-approaches to distinguish epididymal X- and Y-bearing spermatozoa, such existing procedures have not been successful to label oocyte-associated sperm. We describe a FISH method that maintains the integrity of this association and differentiates oocyte-associated X- versus Y-bearing sperm. Cumulus oocyte complexes (COCs) were collected from NIH Swiss females on a control diet. Spermatozoa were obtained from caudal epididymis of male mice, capacitated, and used to inseminate COCs under standard culture conditions. We analyzed the epididymal sperm (n=3961) beforehand, and 50.25 ± 3.6% (n=1987) were Y-sperm and 49.75 ± 3.6% (n=1974) were X-sperm, which are not different from 1:1 ratio. A total of 20 oocytes with 1539 bound sperm were analyzed by a modified XY FISH, as described previously (Whyte et al. 2007), which included incubating slides in 0.01% pepsin/ 0.01N HCl solution for 20 min at 37°C to digest the zona pellucidae and thereby permit the probes to penetrate and bind to sperm DNA. We were able to determine total number of sperm bound to each oocyte and sex chromosome each harbored (Figure 1). The average numbers of X and Y spermatozoa per oocyte were 40.7 ± 4.2 and 36.3 ± 3.8, respectively. These values were not statistically different from 1:1. An average of 77.0 ± 7.8 total sperm associated with each oocyte. This FISH procedure provides a valid and effective way to assess ratios of X and Y sperm associated with murine oocytes. Additionally, as no difference in X to Y sperm ratios was observed, the data dispute the hypothesis that murine oocytes selectively attract one class of sperm over the other in normal mice. It is plausible though that maternal diet or other factors alters expression of zona pellucida proteins or chemoattractants that the oocyte might produce to govern which class of sperm bind and fertilize it. This modified XY FISH procedure provides the groundwork for our future studies to analyze whether contrasting maternal diets with varying fat content alters ability of oocytes to recruit and bind one class of sperm over another. Figure 1a and b Representative images of X- and Y-sperm associated with oocytes (dotted circles) after 5 h in vitro fertilization. X-sperm are stained in green and Y-sperm are stained in red, and the nuclei are in blue (DAPI)