Human sperm mRNA: differential expression between fertile and infertile men and maintenance of transcripts after fertilization

CONRADO AVENDAÑO; ANAHÍ FRANCHI; SERGIO OEHNINGER

San Francisco, CA, USA

Congreso; American Society for Reproductive Medicine 64th Annual Meeting; 2008

American Society for Reproductive Medicine

OBJECTIVE: To evaluate the expression levels of the selected mRNAs: protamine 2 (PRM2), pregnancy specific beta 1-glycoprotein 1 (PSG1) and the nonclassical histocompatibility class I antigen (HLA-E) in ejaculated spermatozoa from fertile and infertile men, and to analyze the fate of these mRNA molecules following microinjection of a single human spermatozoon into hamster oocytes. DESIGN: Prospective study. MATERIALS AND METHODS: 31 infertile patients with diagnostic of male factor or idiopathic infertility and 11 fertile donors were studied. In order to recover a purified population of highly motile spermatozoa, free of somatic and germ cell contaminants, a swim up separation was performed. A total of 1  106 sperm per sample were used for RNA extraction. Real time RT-PCR was performed for mRNA quantification using the housekeeping gene beta-actin (ACTB) to obtain normalized values. Protein expression was examined by immunoblotting. After human sperm microinjection, hamster oocytes were incubated in development medium for 0, 3, 6, 12 or 24 hours. RNA extraction and RT-PCR was done for each individual zygote.  106 sperm per sample were used for RNA extraction. Real time RT-PCR was performed for mRNA quantification using the housekeeping gene beta-actin (ACTB) to obtain normalized values. Protein expression was examined by immunoblotting. After human sperm microinjection, hamster oocytes were incubated in development medium for 0, 3, 6, 12 or 24 hours. RNA extraction and RT-PCR was done for each individual zygote. RESULTS: PRM2, PSG1 and HLA-E were detected by RT-PCR in all groups. PRM2 levels were no significant different between fertile and infertile groups (0.39 ± 0.12 and 0.96 ± 0.66, respectively, p > 0.05). However, PSG1 and HLA-E levels were significantly different between groups (PSG1:2.81 ± 1.39 and 0.53 ± 0.08; and HLA-E: 6.36 ± 4.81 and 0.59 ± 0.12, p <0.05), but the proteins coded by these two transcripts were undetectable. After ICSI, PRM2 transcript was not detected in the hamster oocyte before or at any time after fertilization. However, PSG1 and HLA-E mRNA were detected at all times of zygotic development after injection. CONCLUSIONS: PSG1 and HLA-E mRNA were present at higher levels in fertile than in infertile individuals, while PRM2 mRNA, without a probable function in the fertilized oocyte (and even a deleterious one), did not differ among groups. These findings, in addition to the selected persistence of sperm PSG1 and HLA-E mRNA but not PRM2 in the egg after fertilization, strongly support the concept that the delivery of PSG1 and HLA-E transcripts by the sperm could have a function in early embryo development.