367 FLUORESCENT IN SITU HYBRIDIZATION FOR SEX DETERMINATION AT PROGRESSIVE STAGES OF FERTILIZATION AND DEVELOPMENT IN MICE

Abstract

In mice, relative numbers of male and female pups in a litter can not only vary but may change over the course of pregnancy in response to a range of environmental and physiological factors. The ability to determine the temporal nature of changes in gender ratios in mice has extrapolative value for agriculture, endangered species, and human applications. We have developed a robust and accurate fluorescent in situ hybridization (FISH) suite for visualization of X and Y chromosomes in sperm, pre- and post-implantation conceptuses, and dead/partially cannibalized pups with minimal modification for various samples. Epididymal sperm was collected from NIH Swiss males (n = 5; 15 weeks old) and prepared for FISH by using a modified microwave decondensation–denaturation technique. Pre-implantation embryos (0.5 dpc) collected from pregnant females (n = 9; 20 weeks old) were cultured to the morula stage to permit the development of several blastomeres for FISH analysis. They were then acid-treated to remove the zona pellucida and allowed to adhere to a single slide. Two pre-hybridization procedures, microwave decondensation–denaturation and pepsin digestion (100 µg mL^-1 in 0.01 N HCl), were compared for efficacy. Cranial tissue homogenates in phosphate-buffered saline from post-implantation conceptuses (8.5 dpc) collected from pregnant females (n = 8; 20 weeks old) were fixed to pre-etched slides. To determine whether this procedure could be used on partially cannibalized or stillborn pups, 2 such litters with n = 7 pups from each were retrieved from NIH Swiss females. As the dams had cannibalized the caudal half of the pups, the sex of these could not be determined based on anogenital distance. A small sample of tissue from the remaining cranial region from each was prepared as above for XY FISH analysis, allowing genotypic sexing in pups that would otherwise be of undetermined gender. For each stage of development, slides were hybridized overnight with identical commercially available fluorescence-labeled DNA probes for the X (FITC) and Y (Cy3) chromosomes. Epididymal sperm ratios met the expected value of 0.5. Pepsin treatment of pre implantation embryos provided superior results over microwave decondensation with distinct fluorescence of X and Y chromosomes in morula. Mean sex ratios of 0.5 were determined for both 0.5 and 8.5 dpc embryos (n = 11 ± 1 and 10 ± 1 embryos per female, respectively). Sex determination in post-implantation embryos was confirmed by using 2 separate PCR analyses. The procedure was also successful in recovered postmortem pups and yielded 8 females and 6 males between the 2 litters. This technique may thus be used to detect selective post-natal mortality or cannibalism of one gender over the other. FISH analysis of sperm and multiple pre- or post-implantational embryos or postmortem pups provides a cost-effective and more accurate alternative to PCR-based sex determination. This technique may have potential for use in studies where offspring sex ratios have been influenced by external factors, but wherein the mechanism and stage at which the skewing occurs is uncertain.