Cristina Joana Marques*, Bruno Vaz*, Paula Costa*, Sónia Sousa†, Filipa Carvalho*, Susana Fernandes*, Joaquina Silva†, Mário Sousa†‡, Alberto Barros†

*Serviço de Genética, Faculdade de Medicina da Universidade do Porto; †Centro de Genética da Reprodução Prof. Alberto Barros; ‡Laboratório de Biologia Celular, Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto

Resumo

Introdução: Efectuamos um estudo de pacientes com oligozoospermia e azoospermia para determinar se o imprinting genómico dos espermatozóides se encontra alterado. Métodos: Analisaram-se 23 amostras de sémen de pacientes em estudo de infertilidade conjugal, 7 com normozoospermia (controlos) e 16 com oligozoospermia (9 moderada; 7 severa); e 7 pacientes azoospérmicos sujeitos a biópsia testicular para microinjecção intracitoplasmática de espermatozóide, 3 com espermatogénese conservada (controlos: 2 anejaculação, 1 azoospermia obstrutiva secundária) e 4 com hipoespermatogénese (HP). O DNA dos espermatozóides foi descondensado, purificado e sujeito a mutagénese dirigida por tratamento com bissulfito de sódio. Amplificou-se por PCR (“Polymerase Chain Reaction”) a região diferencialmente metilada (18 CpGs) do gene H19 (metilação paterna), incluindo o local-6 de ligação da proteína CTCF (“parental-allele specific CCCTC-binding factor”) que é igualmente responsável pelo controlo da expressão do gene IGF2 (“Insulin-like Growth Factor 2”). Os fragmentos de PCR foram clonados em plasmídeos e o estado de metilação de cada citosina foi determinado por sequenciação automática. Resultados: Verificou-se uma proporção significativamente mais elevada de: 1) hipometilação global do H19 na HP (p=0,001), 2) atingimento de ≥3 CpGs na oligozoospermia severa e HP (p<0,001) e 3) de todas as 18 CpGs na HP (p<0,001). Em relação ao local-6 de ligação da CTCF, observou-se uma proporção significativamente mais elevada de 1) hipometilação de ≥3 CpGs na HP (p<0,001) e 2) de todas as 5 CpGs na HP (p<0,001). Conclusões: O estabelecimento do imprinting genómico não se encontra conservado na espermatogénese dos pacientes masculinos inférteis com oligozoospermia severa e azoospermia secretora, agravando-se com a severidade da lesão testicular.

Palavras-chave: azoospermia; oligozoospermia; espermatozóides; H19; imprinting genómico; infertilidade masculina.

Abstract

Introduction: We studied infertile patients with oligozoospermia and azoospermia to determine if these conditions of decreased sperm production are associated with defective genomic imprinting. Methods: We included 23 semen samples from men undergoing investigation of infertility as follows: 7 with normozoospermia (controls) and 16 with oligozoospermia (9 moderate; 7 severe); and 7 testicular biopsies, before intracytoplasmic sperm injection: 3 with conserved spermatogenesis (controls: 2 anejaculation, 1 secondary obstructive azoospermia) and 4 with hypospermatogenesis (HP). Sperm DNA was decondensed, purified and treated with sodium bisulfite. The differential methylated region (18 CpGs) of the H19 gene (paternally methylated) was amplified by PCR (Polymerase Chain Reaction), including the CTCF (parental-allele specific CCCTC-binding factor) binding site-6 that also controls the expression of IGF2 (Insulin-like Growth Factor 2) gene. PCR fragments were then cloned in plasmids and the methylation status of each cytosine was determined by automated sequencing. Results: We found a significant higher proportion of 1) global H19 hypomethylation in HP (p=0.001), 2) attainment of ≥3 CpGs in severe oligozoospermia and HP (p<0,001) and 3) of all 18 CpGs in HP (p<0,001). Regarding the CTCF binding site-6, we observed a significant higher proportion of 1) hypomethylation attaining ≥3 CpGs in HP (p<0,001) and 2) all 5 CpGs in HP (p<0,001). Conclusions: The establishment of genomic imprinting is defective in the male germ line of patients with severe oligozoospermia and hypospermatogenesis, being aggravated as the testicular injury worsens.

Key-words: azoospermia; oligozoospermia; genomic imprinting; H19; male infertility; spermatozoa.

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Correspondência:

Dr.ª Cristina Joana Marques

Serviço de Genética Faculdade de Medicina da Universidade do Porto

4200-319 Porto

e-mail: cmarques@med.up.pt