PARENT SESSION

(T281) ABSENCE OF ACCESSORY SEX GLAND SECRETION PERTURBS THE ACTIVE DNA DEMETHYLATION OF SPERM GENOME IN VIVO IN FERTILIZED OOCYTES OF MOUSE AND GOLDEN HAMSTER.

Poon, Hong-Kit¹, O, Wai-Sum², Ferguson-Smith, Anne³, Chow, Pat-Ham¹, ¹ The Chinese Unversity of Hong Kong, Hong Kong SAR² Hong Kong University, Hong Kong SAR³ University of Cambridge, Cambridge, UK

ABSTRACT- Our previous in vivo studies in rodents have shown a beneficial effect of accessory sex gland (ASG) on normal development of both pre- and post-implantation embryos without altering the fertilization rate. It has been recently proposed that the global hypomethylation of human sperm DNA impaired the pregnancy rate after IVF without lowering the fertilization rate, which is partly concordant to our previous findings. In most mammals, the male pronuclei undergo active DNA demethylation from 6 h after fertilization. The aim of this study was to determine if ASG secretions which forms seminal plasma alters DNA methylation status of sperm genome. Either all male ASG or only the ventral prostate was surgically removed in male golden hamsters and designated as TX (n=7) or VPX (n=7) group respectively. The sham-operated control group designated as SH (n=7). Similarly, two groups, VPX (n=10) and SH (n=10), were established in mouse model of ICR strain. After mating with operated male, female was sacrificed either at 1 h post coitus (p.c.) to collect the ejaculated sperm in uterus and oviduct, or 6 h p.c. to recover the oocytes. Cauda epididymal sperm from operated males were also collected. The samples were fixed on slides and the global DNA methylation status was investigated by 5-methylcytosine antibody and revealed by fluorescein (FITC). The images were captured by confocal microscope, and the level of methylation in sperm head, male and female pronuclei were quantified by computer software (MetaMorph). Methylation index (ratio of intensities in male and female pronuclei, mpn/fpn) was calculated. No significant difference in methylation levels was observed in all sperm samples. At 6 h p.c., methylation index was higher in experimental groups than sham group of hamster (SH vs TX, P < 0.01) and mouse (SH vs VPX, P < 0.05). Our results suggest that ASG secretion may play a role in active DNA demethylation in male pronuclei after fertilization. This may affect reprogramming of the genome, contributing to significant loss of embryos. The work described in this paper was fully supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region. (Project no. CUHK4419/03 Medicine).

KEY WORDS: accessory sex gland, dna methylation, pronuclei, sperm