Flow cytometric sorting of non-human primate sperm nuclei.
Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets (Callithrix jacchus), seven hamadryas baboons (Papio hamadryas) and two common chimpanzees (Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 +/- 0.03, 4.20 +/- 0.03, 3.30 +/- 0.01, and 2.97 +/- 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 +/- 0.9 and 93 +/- 0.8%, 94 +/- 0.7 and 94 +/- 0.5%, 91 +/- 0.9 and 97 +/- 0.6%, 94 +/- 0.6 and 94 +/- 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.
O'Brien
Justine K
JK
Faculty of Veterinary Science, Centre for Advanced Technologies in Animal Genetics and Reproduction, University of Sydney, NSW 2006, Australia. justineo@vetsci.usyd.edu.au
Stojanov
Tomas
T
Heffernan
Scott J
SJ
Hollinshead
Fiona K
FK
Vogelnest
Larry
L
Maxwell
W M Chis
WM
Evans
Gareth
G
D000818:Animals; D001562:Benzimidazoles; D002144:Callithrix; D002467:Cell Nucleus / Q000737:chemistry*; D002469:Cell Separation / Q000379:methods / Q000662:veterinary*; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D005456:Fluorescent Dyes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000662:veterinary; D008297:Male; D002679:Pan troglodytes; D048529:Papio hamadryas; D011323:Primates; D012732:Sex Determination Analysis / Q000662:veterinary*; D013094:Spermatozoa / Q000648:ultrastructure*; D014960:X Chromosome; D014998:Y Chromosome
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride