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ABSTRACT: Sperm protamine deficiency has been associated with human male infertility. However, most studies have adopted a global approach to assessing sperm protamine levels. Thus, it is not known whether sperm cells from individual human males possess variations in protamine protein content. The objectives of this study were to evaluate variations in protamine-1 (P1) and protamine-2 (P2) content between individual sperm cells of fertile and infertile men and to correlate DNA integrity and sperm cell viability with protamine levels in individual sperm cells. The semen samples of fertile and infertile men were evaluated globally for protamine protein content using nuclear protein extraction, gel electrophoresis, and densitometry analysis. Individual sperm cell P1 and P2 levels were assessed using immunofluorescence microscopy in conjunction with automated image analysis. The terminal transferase dUTP nick end labeling (TUNEL) assay was performed simultaneously with protamine immunostaining to assess the relationship between protamine levels and DNA integrity in individual spermatozoa. Additionally, the relationship between sperm cell viability and protamine levels was assessed via viability staining concomitant with protamine staining. The protamine fluorescence data demonstrate significant variations in protamine content within individual sperm cells of human males. Overall population-based measures of DNA integrity and sperm cell viability correlate significantly with population-based measurements of protamine levels. The data also demonstrate individual sperm cells displaying the lowest protamine levels display diminished viability and increased sperm cell susceptibility to DNA damage.
During spermiogenesis the protamine proteins replace the somatic cell histones, a process that results in a highly condensed transcriptionally silent chromatin (Oliva and Dixon, 1991; Aoki and Carrell, 2003). In humans, there are 2 protamine proteins, protamine-1 (P1) and protamine-2 (P2), which occur in a strictly regulated one-to-one ratio (Corzett et al, 2002).
Aberrations in protamine expression have been associated with male infertility (Chevaillier et al, 1987; Balhorn et al, 1988; Chevaillier et al, 1990; Belokopytova et al, 1993; de Yebra et al, 1993; de Yebra et al, 1998; Carrell and Liu, 2001; Aoki et al, 2005,2005). A number of studies have described infertile male populations with abnormally elevated ratios of P1 to P2 (P1/P2) (Balhorn et al, 1988; Chevaillier et al, 1990; de Yebra et al, 1993; de Yebra et al, 1998; Carrell and Liu, 2001; Aoki et al, 2005,2005). Two of these reports document a small population of infertile men with complete selective absence of P2 (de Yebra et al, 1993; Carrell and Liu, 2001). Recently, another population of infertile males was identified with deregulated P1 expression and abnormally reduced P1/P2 ratios (Aoki et al, 2005,2005). Taken together, these studies indicate that abnormal protamine stoichiometry derives from aberrant expression of either P1 or P2.
Human sperm protamine deficiency correlates significantly with diminished semen quality parameters, sperm functional ability, and sperm DNA integrity (de Yebra et al, 1993; de Yebra et al, 1998; Balhorn et al, 1999; Carrell and Liu, 2001; Aoki et al, 2005,2005). Due to diminished sperm counts, motility, head morphology, and sperm penetration assay scores (SPA), the majority of male infertility patients with sperm protamine deficiency are treated using in vitro fertilization (IVF) in conjunction with intracytoplasmic sperm injection (ICSI).
Mouse knockout models clearly demonstrate that sperm protamine haploinsufficiency directly impairs spermatogenesis and subsequent embryo development (Cho et al, 2001; Cho et al, 2003). However, human IVF/ICSI outcomes have diverged from these data, not yet resolving a relationship between sperm protamine deficiency and embryonic development or pregnancy rates (Carrell and Liu, 2001; Aoki and Carrell, 2003; Nasr-Esfahani et al, 2004; Aoki et al, 2005,2005). Given these data, it appears ICSI is able to overcome the diminished semen quality and sperm functional ability associated with sperm protamine deficiency in humans.
The majority of studies describing human sperm protamine content have utilized global assessments of protamine levels in whole ejaculates. Thus, it is not known whether sperm cells from individual human males possess variations in protamine levels. The objectives of this study were to evaluate variations in P1 and P2 content between individual sperm cells of fertile and infertile men and to correlate those protamine levels with the DNA integrity status and viability in individual sperm cells.
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This is the first study to quantitatively assess protamine variations in individual sperm cells from fertile men and infertility patients with and without protamine deficiency. The data suggest that significant variations in P1 and P2 levels exist within sperm populations from individual men. These protamine levels significantly relate to the susceptibility of sperm cells to DNA damage induction and overall sperm cell viability.
During spermiogenesis, the nuclear chromatin undergoes significant remodeling (Hecht, 1988; Hecht, 1998; Fuentes-Mascorro et al, 2000; Aoki and Carrell, 2003; Dadoune, 2003). The protamine proteins facilitate these nuclear changes by replacing the somatic cell histones in a 2-step process (Fuentes-Mascorro et al, 2000). In this study, the nuclear protein replacement sequence was elegantly demonstrated via protamine and transition protein immunofluorescence microscopy of human testicular cells. The first step occurs during the round-spermatid stage and involves replacement of the histones with the transition proteins. Later, during the elongating stage of spermatogenesis, the protamine proteins replace these transition proteins. An interesting aspect of these data is that the transition proteins do not appear to be retained in mature spermatozoa, even in patients diagnosed with sperm protamine deficiency.
The intrasample protamine heterogeneity observed in this study is consistent with other reports using chromomycin A3 (CMA3) and aniline blue staining to indirectly measure protaminization of human sperm cells (Manicardi et al, 1995; Hammadeh et al, 2001). Aniline blue selectively stains lysine-rich histone proteins, leaving the arginine-rich protamine proteins unstained (Hammadeh et al, 2001). CMA3 directly competes with protamine for access to the DNA (Manicardi et al, 1995). Thus, high levels of protamine relate to low levels of aniline blue or CMA3 staining. Results of these indirect staining experiments indicate that, within individuals, protamine levels may vary between individual sperm cells, exemplified by variations in aniline blue and CMA3 staining within sperm cell populations (Bianchi et al, 1993; Manicardi et al, 1995; Manicardi et al, 1998; Hammadeh et al, 2001).
Intrasample protamine variability is of particular clinical significance for assisted reproductive techniques. Patients with protamine deficiency typically present with significantly diminished semen quality parameters, sperm functional ability, and sperm DNA integrity (de Yebra et al, 1993; de Yebra et al, 1998; Balhorn et al, 1999; Carrell and Liu, 2001; Aoki et al, 2005,2005). Due to diminished counts, motility, head morphology, and SPA scores, the majority of these infertility patients are treated using IVF in conjunction with ICSI (Aoki and Carrell, 2003; Aoki et al, 2005,2005).
Protamine-deficient patients undergoing human IVF/ICSI treatment have shown normal embryo quality, implantation, and pregnancy rates (Carrell and Liu, 2001; Nasr-Esfahani et al, 2004; Aoki et al, 2005,2005). These data are inconsistent with animal studies showing that mouse protamine haploinsufficiency directly impairs spermatogenesis and subsequent embryo development (Cho et al, 2001; Cho et al, 2003). However, in these mouse models the protamine deficiency was reported to be homogenous throughout the sperm cell population.
The heterogeneity in protamine levels reported in this study may serve to clarify how ICSI is able to overcome the diminished semen quality and sperm functional ability associated with sperm protamine deficiency in humans. The data indicate that, in patients diagnosed with protamine deficiency via global assessments, a small population of sperm cells is present with normal protamine content, which may be selected for injection during IVF/ICSI. Zhang et al (2006) have recently confirmed that semen samples with a low protamine level contain increased levels of histone 2B, and have confirmed their results using immunohistochemistry analysis of individual sperm. In their study, intrasample variation was observed, similar to our data, indicating focal regions of abnormal spermatogenesis. The sperm cell preparation techniques used in conjunction with IVF/ICSI, such as density gradient centrifugation, may be able to increase the relative concentrations of sperm cells with normal protamine content (Colleu et al, 1996; Sakkas et al, 2000). Additionally, the data indicate compromised viability in protamine-deficient sperm, which may further aid in positive sperm selection during ICSI. Further studies should evaluate protamine levels in fresh vs prepared semen samples. In addition, it will be important to evaluate variations in the P1/P2 ratio between individual sperm cells.
The data also suggest DNA damage is compromised in protamine-deficient human sperm. These results are consistent with other reports which document a relationship between sperm protamine levels and DNA integrity (Bianchi et al, 1993; Manicardi et al, 1995; Manicardi et al, 1998; Aoki et al, 2005,2005). Elegant studies in mice demonstrate protamine haploinsufficiency leads to increases in sperm cell DNA damage (Cho et al, 2003). In the present study, sperm cells with the lowest protamine levels were shown to have significantly increased DNA damage within individuals. Meanwhile, sperm cells with the highest protamine levels demonstrated significantly reduced DNA damage as measured by the TUNEL assay.
These results suggest that normally expressed sperm protamines may serve a protective function against DNA damage. Protamine-deficient sperm appear to be more susceptible to DNA strand breaks, evidenced by the significantly increased DNA damage in sperm cells with the lowest levels of protamine proteins. Sperm with low protamine levels retain higher levels of histone 2B, which may be less effective in protecting sperm DNA from damage (Zhang et al, 2006). Because a mild sperm chromatin decondensation protocol was used in this study, it is likely there was an artificial induction of DNA strand breaks. Indeed, DNA damage was slightly elevated postdecondensation. However, this treatment-induced increase in DNA damage was nonsignificant and comparable between all comparison groups, indicating that decondensation did not bias the DNA damage comparison between cases and controls.
To conclude, this is the first study to demonstrate that significant variations exist in the protamine levels of individual human sperm cells. These protamine variations relate significantly to sperm cell viability and DNA damage and may be of clinical significance, since infertile human males diagnosed with protamine deficiency via global assessments may possess a small population of cells with normal protamine content.