Sperm Nuclear Histone to Protamine Ratio in Fertile and Infertile Men: Evidence of Heterogeneous Subpopulations of Spermatozoa in the Ejaculate

Abstract

Sperm protamine deficiency is observed in a subset of infertile men, suggesting that the relative histone to protamine ratio may be altered in the spermatozoa of these men. We measured the ratio of nuclear histones to protamines in the spermatozoa of fertile (n 5 10) and infertile men (n 5 20). Sperm nuclear proteins were extracted and subsequently separated by acid-urea (AU) polyacrylamide gel electrophoresis. The relative histone (H2B) to protamine (PRM1 + PRM2) and PRM1 to PRM2 ratios were estimated by densitometric analysis of the AU gels. Immunoblotting experiments (using H2B, PRM1, and PRM2 antibodies) were conducted to confirm the specificity of the bands. The pattern and intensity of H2B staining in human spermatozoa was assessed by immunocytochemistry. Sperm samples from the infertile men in this study had a significantly higher proportion of histone H2B to protamine (PRM1 + PRM2) than did samples from the fertile men in this study (0.38 vs 0.08, P , .001). Immunocytochemistry experiments demonstrated a punctuated staining pattern (with strong, intermediate, or weak H2B staining intensity) throughout the sperm head. Infertile men had a higher proportion of spermatozoa exhibiting strong and intermediate staining than did samples from fertile men. These findings suggest that infertile men possess a higher proportion of spermatozoa with an increased histone to protamine ratio than fertile controls.

Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility

“…Scattering of the immunofluorescence signals from histones displayed a punctuated variation in the sperm head (Fig. 4); this pattern was also similar to that reported in previous experiments in swines (Flores et al 2011) and humans (Zhang et al 2006). Additionally, one of the few immunocytochemical studies conducted on bull sperm chromatin has demonstrated that histones are also located in the perinuclear theca (Tovich & Oko 2003).…”

Testis/sperm-specific histone 2B in the sperm of donors and subfertile patients: variability and relation to chromatin packaging

“…Sperm from infertility patients often exhibit anomalies related to the protamine content and composition (Balhorn et al, 1988;Foresta et al, 1992;Belokopytova et al, 1993;de Yebra et al, 1993;Carrell and Liu, 2001;Nasr-Esfahani et al, 2004;Aoki et al, 2005;Oliva, 2006;Torregrosa et al, in press). In addition, an increased ratio between histones and protamines has been reported to exist in the sperm of infertile men (Silvestroni et al, 1976;Terquem and Dadoune, 1983;Juelin et al, 1986;Chevaillier et al, 1987;Auger et al, 1990;Bach et al, 1990Blanchard et al, 1990Zhang et al, 2006). Overall, many of these changes in the basic nuclear protein complement, which presumably make the sperm chromatin more vulnerable, have been shown to correlate with the various nuclear and chromatin disturbances that are manifested in the sperm of many infertile men (Agarwal and Said, 2003).…”

Identification of Genetic Variation in the 5′ and 3′ Non-coding Regions of the Protamine Genes in Patients with Protamine Deregulation

“…The indirect association between abnormal protamine expression and severely altered spermatogenesis may not be intuitively obvious, but animal studies have also shown that spermatogenesis is severely altered when protamine expression is experimentally reduced [Zhong et al 1999;Aoki et al 2005]. In contrast to human data, haploinsufficient mice show a homogenous population of severely affected sperm, while infertile male patients with protamine deregulation exhibit a truly heterogenous population of sperm [Cho et al 2001;Aoki et al 2005;Aoki et al 2006b;Zhang et al 2006]. This may be due to focal disruptions in the tubular structure of the testis or disruption of upstream regulators with an incomplete phenotypic penetrance.…”