Alteração Transmissível do Imprinting Genómico em Pacientes Inférteis por Oligozoospermia e Azoospermia

 

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

Heritable Genomic Imprinting Defects in Infertile Patients With Oligozoospermia and Azoospermia

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.

 

 

Texto completo disponível apenas em PDF.

Full text only available in PDF format.

 

 

REFERÊNCIAS

1 -Surani MAH, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 1984;308:548-50.

        [ Links ]

2 -Arnaud P, Feil R. Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction. Birth Defects Res, part C, Embryo Today 2005;75:81-97.

3 -Kerjean A, Dupont J-M, Vasseur C, et al. Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis. Hum Mol Genet 2000;9:2183-7.

4 -Takai D, Gonzales FA, Tsai YC, Thayer MJ, Jones PA. Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer. Hum Mol Genet 2001;10: 2619-26.

5 -Gosden R, Trasler J, Lucifero D, Faddy M. Rare congenital disorders, imprinted genes, and assisted reproductive technology. Lancet 2003;361:1975-7.

6 -Ludwig M, Katalinic A, Gross S, Sutcliffe A, Varon R, Horsthemke B. Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet 2005;42:289-91.

7 -Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet 2004; 363:1700-2.

8 -WORLD HEALTH ORGANIZATION. (1999). WHO laboratory manual fot the examination of human semen and sperm-cervical mucus interaction (4ª ed.) Cambridge: Cambridge University Press.

9 -Sousa M, Cremades C, Alves C, Silva J, Barros A Developmental potential of human spermatogenic cells cocultured with Sertoli cells. Hum Reprod 2002;17:161-72.

10 -Benchaib M, Ajina M, Lornage J, Niveleau A, Durand P, Guérin JF. Quantitation by image analysis of global DNA methylation in human spermatozoa and its prognostic value in in vitro fertilization: a preliminary study. Fertil Steril 2003;80:947-53.

11 -Lucifero D, Mertineit C, Clarke HJ, Bestor TH, Trasler JM. Methylation dynamics of imprinted genes in mouse germ cells. Genomics 2002;79:530-8.

12 -Li J-Y, Lees-Murdock DJ, Xu G-L, Walsh CP. Timing of establishment of paternal methylation imprints in the mouse. Genomics 2004;84:952-60.

13 -Doerksen T, Benoit G, Trasler JM. Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology. Endocrinology 2000;141:3235-44.

14 -Ariel M, Cedar H, McCarrey J. Developmental changes in methylation of spermatogenesis-specific genes include reprogramming in the epididymis. Nat Genet 1994;7:59-63.

15 -Bliek J, Terhal P, van den Boogaard M-J, et al. Hypomethylation of the H19 gene causes not only Silver-Russell syndrome (SRS) but also isolated asymmetry or an SRS-like phenotype. Am J Hum Genet 2006;78:604-14.

 

 

Correspondência:

Dr.ª Cristina Joana Marques

Serviço de Genética Faculdade de Medicina da Universidade do Porto

Alameda Prof. Hernâni Monteiro

4200-319 Porto

e-mail: cmarques@med.up.pt