Human Reproduction Update Advance Access originally published online on January 5, 2007
Human Reproduction Update 2007 13(3):313-327; doi:10.1093/humupd/dml057
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Altered protamine expression and diminished spermatogenesis: what is the link?
1 Andrology and IVF Laboratories, Department of Surgery 2 Department of Physiology 3 Department of Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, UT, USA
4 To whom correspondence should be addressed at: Andrology and IVF Laboratories, 675 S. Arapeen Dr, Suite #205, Salt Lake City, UT 84108, USA. E-mail: douglas.carrell{at}hsc.utah.edu
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Abstract |
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Abstract
IntroductionDuring the elongating spermatid stage of spermiogenesis, human sperm chromatin undergoes a complex transition in which histones are extensively replaced by protamines in a carefully regulated transition including histone modifications and intermediate and temporary replacement of the histones by sperm-specific transition proteins. The replacement of most histones by protamines 1 and 2 facilitates a high order of chromatin packaging necessary for normal sperm function and may also be necessary for DNA silencing and imprinting changes within the sperm cell. Protamines 1 and 2 are usually expressed in nearly equal quantities, but elevated or diminished protamine 1/protamine 2 ratios are observed in some infertile men and is often associated with severe spermatogenesis defects. Human and animal studies demonstrate that expression of the protamine proteins is uniquely regulated by transcription/translation factors, including storage of the mRNA in ribonucleoprotein (RNP) particles composed of the mRNA, transcription factors and a kinesin molecule necessary for transport of the RNP to the cytoplasm and removal of transcriptional activators from the nucleus. Recent studies indicate that most patients with abnormal protamine protein levels have elevated levels of protamine transcript in the mature sperm cell, indicating a possible defect in transcription or translation. The regulation of protamine expression is unique and includes several possible mechanisms which may be responsible for dysregulation of protamine expression and concurrent broad spectrum defects in spermatogenesis. We suggest two hypotheses: (i) that abnormal protamine expression is indicative of a generalized defect in mRNA storage and/or translation which affects other mRNA transcripts or (ii) that protamines may act as a checkpoint of spermatogenesis.
Key words: chromatin / gene expression / protamine / spermatogenesis / transition protein
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Introduction |
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Sperm chromatin is a highly organized, compact structure consisting of DNA and heterogeneous nucleoproteins. The most abundant nucleoproteins in mature sperm are the protamines, positively charged molecules that replace histones during spermiogenesis. Protamines confer a higher order of DNA packaging in sperm than that found in somatic cells, and the condensed and insoluble nature of the highly condensed sperm chromatin protects the genetic integrity of the paternal genome during its transport through the male and female reproductive tracts (Gatewood et al., 1987
; Balhorn et al., 1999; Brewer et al., 2002). Protamine replacement may also be necessary for silencing of the paternal genome and reprogramming of the imprinting pattern of the gamete (Aoki and Carrell, 2003).
Humans express two protamines, protamine 1 (P1) and protamine 2 (P2), both of which are expressed in roughly equal quantities (Balhorn et al., 1999; Corzett et al., 2002). Protamines are highly basic sperm-specific nuclear proteins that are characterized by an arginine-rich core and cysteine residues (Dixon et al., 1986; Krawetz and Dixon, 1988). The high level of arginine causes a net positive charge that facilitates strong DNA binding (Balhorn et al., 2000). The cysteine residues facilitate the formation of multiple inter and intra-protamine disulphide bonds that are essential for the high order of chromatin packaging necessary for normal sperm function (Courtens and Loir, 1981; Loir and Lanneau, 1984; Singh and Rao, 1988; Le Lannic et al., 1993; Szczygiel and Ward, 2002).
During spermiogenesis, protamines progressively replace somatic histones in a stepwise manner (Dixon et al., 1986). First, somatic histones are replaced by testis-specific histone variants, which are then replaced by transition proteins (TP1 and TP2) in a process that involves extensive DNA rearrangement and remodeling (Ward et al., 1989). During the elongating spermatid stage, the transition proteins are replaced in the condensing chromatin by protamines. In humans, 
85% of the histones are replaced by protamines. (Hecht, 1989, 1990; Oliva and Dixon, 1990; Dadoune, 1995; Steger, 1999). This sequential process facilitates molecular remodelling of the male genome within the differentiating spermatid nucleus (Figure 1) (Sassone-Corsi, 2002).
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Previous studies have shown that the mean P1/P2 ratio in human sperm is approximately 1.0 (Balhorn et al., 1999
; Carrell and Liu, 2001; Oliva, 2006). Sperm from some infertile men have been shown to have altered P1/P2 ratios and/or non-detectable P2 in mature sperm, whereas the occurrence of protamine abnormalities in sperm from fertile men is extremely rare (no known cases have been reported) (Balhorn et al., 1988; Chevaillier et al., 1987; Belokopytova et al., 1993; de Yebra et al., 1998; Aoki et al., 2005a; Oliva, 2006). Additionally, transgenic mice with protamine haploinsufficiency have severely altered spermatogenesis and male infertility (Cho et al., 2001). The link between abnormal protamine levels and infertility is intriguing because abnormal protamine expression has been associated with low sperm counts, decreased sperm motility and morphology, diminished fertilization ability and increased sperm chromatin damage, some of which are not intuitively linked to abnormal chromatin structure (Carrell and Liu, 2001; Mengual et al., 2003; Aoki et al., 2005a). A direct relationship between abnormal protamine expression and sperm count, motility, morphology or fertilization ability is not readily apparent. The reduction in P1 or P2 in these patients may be explained by reduced protamine transcription, altered translation of the transcript or failed post-translational modifications, but none of these scenarios would directly explain the associated decline in sperm counts and function unless the regulation of protamine exchange is linked to a broader control of spermatogenesis.
This review will briefly summarize the current understanding of protamine replacement of histones, the link between altered protamine replacement and male infertility, the regulation of protamine expression during spermatogenesis and possible causes of altered protamine expression. Two possible models will be discussed regarding the link between abnormal protamine expression and aberrant spermatogenesis. The first hypothesis is that abnormal protamine expression is indicative of a general abnormality of spermatogenesis, possibly due to abnormal function of a transcriptional or translational regulator. Candidate regulatory factors will be discussed. The second hypothesis to be discussed is that the protamines may act as a checkpoint regulator of spermatogenesis and that abnormal protamine expression leads to induction of an apoptotic process that ends in severely diminished semen quality.
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Clinical significance of abnormal protamine expression |
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Abnormal protamine expression is clearly associated with infertility, as recently thoroughly reviewed by Oliva (Oliva, 2006
). Briefly, studies have identified males with undetectable P2, which has consistently been linked to severe male infertility (de Yebra et al., 1993; Carrell and Liu, 2001). In mice, haploinsufficiency of the protamines has been shown to cause altered spermatogenesis, including lowered sperm counts and DNA damage (C. Cho et al., 2001; Cho et al., 2003). Numerous studies have demonstrated that an altered ratio of P1/P2, either increased or decreased, is associated with reduced fertility (de Yebra et al., 1998; Carrell and Liu, 2001; Aoki et al., 2006d; Oliva, 2006). Interestingly, no reports have been made of P1/P2 expression abnormalities in males of known fertility.
Initial studies suggested that the most common protamine abnormality in infertile men was an elevated P1/P2 ratio (Oliva, 2006). The elevated P1/P2 ratio is often the result of decreased P2 protein levels, concomitant with an increased level of P2 precursors (Carrell and Liu, 2001; de Yebra et al., 1998; Aoki et al., 2006d). Under expression of P2 accounts for the majority of the cases with high P1/P2 ratio, but subsequent studies have demonstrated that P1 dysregulation also accounts for some abnormalities (Aoki et al., 2005a). However, P2 dysregulation is more common and this may be explained by the fact that the P2 gene is derived more recently than the P1 gene, which may suggest that the regulatory mechanisms governing P2 gene expression are not as stringent and more susceptible to variation than the P1 gene (Lewis et al., 2003).
Human sperm protamine dysregulation is associated with diminished semen quality parameters, sperm functional ability and sperm DNA integrity (de Yebra et al., 1993, 1998; Balhorn et al., 1999; Carrell and Liu, 2001; Aoki et al., 2005a). Aoki et al. (2005b) have shown that sperm concentration, motility and morphology are significantly reduced in patients with either a low or a high P1/P2 ratio when compared with patients with a normal P1/P2 ratio. In addition, an altered P1/P2 ratio is associated with decreased fertilization ability, although fertilization and pregnancy rates are not different when patients undergo intracytoplasmic sperm injection (ICSI) as opposed to standard in vitro fertilization (IVF) (Carrell and Liu, 2001; Nasr-Esfahani et al., 2004; Aoki et al., 2005b).
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Protamines and DNA damage |
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One potential consequence of abnormal protamine expression is a susceptibility to DNA damage. Our laboratory has measured DNA integrity using an assay similar to the sperm chromatin structure assay and compared the DNA fragmentation index with protamine levels in human sperm. Patients with low P1/P2 ratio had significantly elevated DNA fragmentation when compared with patients with normal and high P1/P2 ratios (Aoki et al., 2005b
). Moreover, patients who under-expressed P1, P2 or both P1 and P2 had significantly elevated levels of DNA fragmentation compared with patients normally expressing P1and P2. Additionally, Torregrosa et al. (2006) have recently shown a positive correlation between TUNEL-positive sperm and the presence of P2 precursors. These studies emphasized the important role protamines play in protecting the genetic content of the mature sperm from nucleases.
A recent study evaluated the role of protamine abnormalities at an individual cell level by using fluorescence immunohistochemistry techniques to simultaneously evaluate protamine levels, cell viability and DNA damage as measured by the TUNEL assay (Aoki et al., 2006c). Concurrently, global protamine levels were evaluated with a fraction of the semen sample that underwent standard nuclear protein extraction and electrophoresis. The data not only confirmed a close correlation between the mean protamine levels determined by fluorescence microscopy and the standard electrophoresis technique, but also showed that within a semen sample there is heterogeneity in protamine expression and a clear correlation between under-expression of protamines, DNA damage and lack of viability (Aoki et al., 2006c). The intra-ejaculate protamine heterogeneity observed in this study is consistent with other reports using CMA3 and Aniline Blue staining to assess protamine quantity indirectly (Manicardi et al., 1995; Hammadeh et al., 2001), but novel in the direct link between protamine abnormalities in a given cell and DNA damage within the cell.
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Possible mechanisms of DNA damage |
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DNA nicks may be induced through apoptotic processes (Cisternas and Moreno, 2006
). Apoptosis regulates germ cell over proliferation and eliminates defective germ cells from the genetic pool (Hikim et al., 1995; Rodriguez et al., 1997; Pentikainen et al., 1999). Apoptosis is characterized by DNA double-stranded breaks which occur as a result of activated endogenous DNA nucleases (Gorczyca et al., 1993). In somatic cells, the apoptotic cascade involves the formation of apoptotic body; however, in highly differentiated spermatozoa, the sequence of events may differ as a result of the highly condensed sperm nucleus (Hikim et al., 1995). Spermatozoa that are marked for apoptotic degradation may have normal mitochondrial activity, high or low motility (Barroso et al., 2000) as well as normal morphology (Host et al., 2000a,b). Oosterhuis et al. (2000) reported that 20% of ejaculated spermatozoa showed DNA strand breaks and the apoptotic marker annexin V, whereas, Sakkas et al. (1999) reported that DNA strand breaks and apoptotic markers did not co-exist together in the same mature spermatozoa. Ejaculated spermatozoa with apoptotic markers appeared to have escaped programmed cell death in a process called abortive apoptosis (Sakkas et al., 1999). Therefore, it will be important to distinguish between cells that show high levels of DNA strand breaks and cells that are positive for apoptotic markers. It is inappropriate to assume that strand breaks are synonymous with apoptotic degeneration.
DNA damage may also be increased if the DNA nicking and ligating activities of topoisomerase II are defective. The presence of higher than usual levels of topoisomerase II found during the elongating spermatid stage is associated with high levels of DNA nicks (Roca and Mezquita, 1989; McPherson and Longo, 1993), possibly needed to relieve torsional stress caused by the negative supercoiling associated with histone to protamine transition (Balhorn, 1982; Risley et al., 1986; McPherson and Longo, 1993). These nicks are not usually harmful, since they are usually re-ligated prior to completion of spermiogenesis and ejaculation (McPherson and Longo, 1993). However, if the activity of topoisomerase is blocked or disrupted, then DNA nicks remain in mature sperm or are not repaired properly (Morse-Gaudio and Risley, 1994).
Shaman et al. have recently demonstrated that topoisomerase II likely acts in two ways. First, it relieves torsional stress by causing double strand breaks which are re-ligated (termed sperm chromatin fragmentation). Second, it acts in conjunction with an extracellular nuclease to cause regulated double-strand breaks in protamine-bound DNA at 50 kb intervals, the DNA span of one loop bound to protamine (termed sperm DNA degradation) (Sotolongo et al., 2005; Shaman et al., 2006). In the absence of protamines, extensive degradation occurs. This topoisomerase/nuclease-induced DNA degradation may be a specialized apoptotic pathway in sperm, different from the normal function of topoisomerase in relieving torsional stress, followed by re-ligation of the DNA break (Shaman et al., 2006).
Caron et al. (2001) suggested that the transient DNA nicks can be repaired by transition protein 1. Transition proteins have been found to have an undefined enzymatic activity that is responsible for repairing single-stranded breaks and UV-induced DNA lesions in vivo; therefore, the role of transition proteins extends beyond initiating DNA compaction to restoring transient DNA nicks. Evidence from the literature indicates that the disappearance of single-strand breaks during spermiogenesis is coincident with the presence of the transition proteins in elongating spermatid (Sakkas et al., 1995; Kistler et al., 1996; Smith and Haaf, 1998).
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Reactive oxygen species and chromatin damage |
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In recent years, concern has been expressed about the generation of reactive oxygen species (ROS) in the male reproductive tract. High levels of ROS are toxic to sperm quality and function (Saleh and Agarwal, 2002
). Elevated levels of ROS have been reported in 25–40% of the infertile patients (Padron et al., 1997). Two factors that protect the DNA from oxidative stress are tight DNA packaging and antioxidants present in seminal plasma (Twigg et al., 1998). Oxidative stress happens as a result of the imbalance between ROS generation and antioxidants scavenging activities (Sikka, 2001). Strong evidence suggests that the presence of single and double-stranded breaks observed in infertile patients is a result of ROS (Fraga et al., 1996; Kodama et al., 1997; Sun et al., 1997; Aitken and Baker, 2004). The presence of 8-hydroxy-2-deoxyguanosine in seminal plasma has been used as a marker for oxidative DNA damage (Ames et al., 1993). A significant positive correlation was established between DNA fragmentation and ROS (Barroso et al., 2000). Furthermore, exposure of sperm to artificially produced ROS resulted in a significant increase in DNA damage in the form of deletions, frame shifts, DNA cross-links and chromosomal rearrangements (Twigg et al., 1998a,b; Kemal Duru et al., 2000). However, direct studies on ROS-induced damage in protamine deficient sperm have not been performed. |
Protamine abnormalities and assisted reproduction techniques |
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The intrasample heterogeneity of protamine content is clinically significant for patients undergoing assisted reproductive technology (ART) (Aoki et al., 2006c
). Protamine-deficient patients undergoing human IVF/ICSI have been shown to have normal embryo quality, implantation and pregnancy rates (Carrell and Liu, 2001; Nasr-Esfahani et al., 2004; Aoki et al., 2005b). This heterogeneity in protamine concentration in sperm of a given semen sample may explain how ICSI appears to overcome poor semen quality and DNA damage (Aoki et al., 2006e). It is possible that selection of the most morphologically normal motile sperm for ICSI injection inherently selects for sperm with normal protamine expression, although studies evaluating sperm morphology and protamines in individual sperm have not been performed. Interestingly, the above findings are inconsistent with the data presented from the mouse protamine-deficient haploinsufficiency model, in which haploinsufficient mice were found to have a higher rate of embryo death when ICSI was performed (Cho et al., 2001, 2003). This difference in embryo lethality, reflecting a more severe effect of abnormal protamine expression in this model, may be the result of a homogeneous pathology throughout the seminiferous tubule rather than the variable expression seen in infertile men with protamine expression defects or may be the result of a lack of effect on sperm morphology in mice.
The long-term consequences of ICSI with DNA-damaged sperm is still not clear (Silber, 1995; Ludwig, 2005; Verpoest and Tournaye, 2006). Animal studies have suggested a sperm DNA damage threshold below which a normal embryo can develop (Ahmadi and Ng, 1999). Others have demonstrated that the oocyte may have the DNA repair system that aids in ‘repairing’ altered chromatin (Ashwood-Smith and Edwards, 1996; Perry et al., 1999). Another concern regarding the use of sperm with abnormal chromatin is the potential for improper gene imprinting, since protamines have been suggested to be a possible regulator of normal genomic imprinting (Aoki and Carrell, 2003; Oliva, 2006) and since imprinting errors have been suggested to be elevated in patients undergoing ART (Allen and Reardon, 2005; Chang et al., 2005; Ludwig et al., 2005). Hartman et al. (2006) have recently noted no increase in imprinting errors in men with severe spermatogenesis defects. Our laboratory has recently reported that defects in global methylation are not observed in men with known protamine abnormalities (Aoki et al., 2006b). Clearly, there is a need for further studies to evaluate specific gene imprinting in those patients and other potential defects in sperm with abnormal protamine replacement and/or DNA damage.
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Protamine replacement of histones |
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Although protamine replacement is often termed ‘a two-step process’ (histones replaced by transition proteins which are replaced by protamines), more steps are involved, including the expression and incorporation of testis-specific histone variants, histone hyperacetylation, replacement of histones with transition proteins and protamine incorporation and phosphorylation (Aoki and Carrell, 2003
; Churikov et al., 2004b). Each of these steps is critical to proper progression of chromatin maturation and spermiogenesis. |
Histone modifications |
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In somatic cells, nucleosomes are composed of two molecules of histones 2A, 2B, 3 and 4 (H2A, H2B, H3 and H4). Histone 1 (H1 or linker histone) links inter-nucleosomal DNA. In addition to the somatic-type histone variants, spermatogenic cells express testis-specific histones that replace somatic histones (Dadoune, 2003
). Although termed ‘testis-specific’ histones, at least one testis variant, an H3 variant, has been shown to be expressed in somatic cells (Govin et al., 2005).
The characterization of testis-specific histone variants is in the early stages, but several testicular variants have been identified in the human for both the nucleosome and linker histones (Churikov et al., 2004b; Govin et al., 2005). The differences observed between testis-specific histones and somatic histones include structural differences in the N-terminal region, the core region and the C-terminal region. Interestingly, some testis-specific histone variants do not undergo 3' polyadenylation and are translated early during spermatogenesis (Zalensky et al., 2002; Churikov et al., 2004a). Among those variants is an H2B variant which has been shown to localize in telomeres and may be important in meiosis (Gineitis et al., 2000). Another key difference in the testis variant of H2B is the replacement of four prolines found in the N-terminal region of the somatic H2B with phosphorylatable amino acids, likely indicating that their function is regulated by phosphorylation (Churikov et al., 2004a).
Recently, Zhang et al. (2006) have shown that increased levels of histone 2B in sperm is associated with lower levels of protamines. Although previous studies have demonstrated high levels of histones in the sperm of some infertility patients and an indirect link between histone retention and altered sperm protamine expression, this study is the first direct evidence of abnormal histone retention linked to altered protamine replacement. Future studies will likely focus on the further characterization of histone variants and whether abnormal expression of a testis-specific histone variant may be directly responsible for altered protamine replacement. In that regard, Tanaka et al. have recently evaluated the gene sequence of HANP1 in infertile and fertile men. HANP1 is the human orthologue of the mouse Hanp1/Hit2 gene that encodes a testes variant of H1; homozygous disruptions of this gene in mice has previously been shown to cause male infertilty (Tanaka et al., 2005, 2006). Although five single nucleotide polymorphisms (SNPs) were identified for HANP1 in their study population, the SNPs did not appear to be linked to male infertility (Tanaka et al., 2006). Further studies are warranted to evaluate both the protein and the gene in males with known protamine abnormalities.
Hyperacetylation of the histones is critical for normal progression of spermatogenesis and is regulated by an interplay of histone acetyl transferases and histone deacetylases (Candido and Dixon, 1972; Grimes and Henderson, 1984; Meistrich et al., 1992; Hazzouri et al., 2000; Marcon and Boissonneault, 2004). Histone hyperacetylation reduces the binding between nucleosomes and DNA, leading to chromatin relaxation (Hong et al., 1993), and is also associated with the activation of topoisomerases in inducing strand breaks. Species that retain histones throughout spermiogenesis have relatively low levels of acetylated histones (Kennedy and Davies, 1980, 1981). It has been suggested that hyperacetylation of core histones may facilitate their displacement by protamines (Oliva and Mezquita, 1982, 1986; Oliva et al., 1987), and a double bromodomain containing testis-specific factor (BRDT) has been identified in mice as a possible key factor in the transition process. BRDT has been shown to be capable of condensing acetylated chromatin (Pivot-Pajot et al., 2003) by recruiting a highly expressed chaperone protein, CIA-II, to mediate histone removal (Umehara and Horikoshi, 2003) .
Sonnack et al. (2002) have demonstrated a relationship between decreased acetylation and abnormal spermatogenesis. They also observed increased acetylation in spermatocytes of testes exhibiting maturation arrest, indicating a possible relationship between premature hyperacetylation and maturation arrest. This same laboratory has also demonstrated that the administration of histone deacetylases results in severe infertility (Fenic et al., 2004). Future studies will likely evaluate the degree of acetylation in protamine-deficient patients and experimental models with abnormal protamine expression.
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Replacement of histones with transition proteins |
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DNA relaxation, as a result of hyperacetylation and topoisomerase activity, facilitates the exchange of histones with transition proteins which are proteins of intermediate basicity (Wilkins, 1956
; Courtens and Loir, 1981; Luerssen et al., 1989; Oliva and Dixon, 1991; Ward and Coffey, 1991; Kierszenbaum, 2001; Meistrich et al., 2003). TP1 and TP2 mRNA are first seen in the post-meiotic, round spermatid stage in mice and are degraded around stages, 13–14. The proteins are observed in stages 12 and 13 and are removed by stage 14 when they are replaced with protamines (Figure 2) (Kistler et al., 1996). There is significant overlap in the expression of histones, transition proteins and protamines (Meistrich et al., 2003). Expression of the proteins has been shown to have some overlap in human-elongating spermatids (Aoki et al., 2006c).
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TP1 is a 60 kDa protein with numerous basic amino acids distributed randomly throughout the molecule (Kistler et al., 1975
). TP1 has important DNA-destabilizing properties due to the presence of two tyrosine residues flanked by basic amino acids (Singh and Rao, 1988). TP2 is a 13 kDa protein that contains proline, serine, arginine and lysine residues (Grimes et al., 1975). Two potential zinc finger domains exist in mouse and rat TP2 and may play an important role in the initiation of chromatin condensation and cessation of transcriptional activity during mammalian spermiogenesis (Baskaran and Rao, 1991). In transgenic mice, animals devoid of transition protein 2 had reduced amounts of processed P2 proteins and failed to complete chromatin compaction (Cho et al., 2003; Meistrich et al., 2003). In addition to incomplete chromatin condensation, sperm from TP2 null mice show an increase in DNA denaturation when compared with sperm from control mice (Zhao et al., 2001). The increased denaturability of the DNA is believed to result from DNA strand breaks (Sailer et al., 1995; Aravindan et al., 1997).
Studies using double knock out mice for both TP1 and TP2 have shown that the absence of one transition protein does not affect the level of transcription or translation of the other transition protein or the protamines, but does affect the retention of the other transition protein through post-translational modifications (Shirley et al., 2004; Zhao et al., 2004). Although the redundancy is not complete, there is compensation for one transition protein by the other, as demonstrated by the fact that double heterozygous mice exhibit more severe sperm defects than do mice homozygous for a single mutation. Interestingly, sperm from transition protein-deficient mice are able to fertilize oocytes using ICSI if the sperm were isolated from the testis or caput epididymus, but are not capable of fertilization if isolated from the cauda epididymus (Suganuma et al., 2005).
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Replacement of transition proteins with protamines |
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Protamine 1 is translated as a mature protein of 50 amino acids, whereas protamine 2 is initially 103 amino acids and undergoes N-terminus cleavage to a mature protein of 57 amino acids (Figure 3) (Aoki and Carrell, 2003
). Following translation, protamine 1 is immediately phosphorylated, primarily under the control of serine/arginine protein-specific kinase 1 (SRPK1) (Green et al., 1994; Papoutsopoulou et al., 1999). A protamine 2 intermediate protein is phosphorylated, largely under the control of Ca2+/calmodulin-dependent protein kinase 4 (CAMK4). The phosphorylation of the P2 intermediate is requisite for binding of the protein to chromatin, which is required for final cleavage of the protein to its mature form of 57 amino acids.
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Protamine phosphorylation is not only necessary for final processing of P2, but also for proper binding of the proteins to DNA. However, once bound to DNA, the protamines are de-phosphorylated. The de-phosphorylation appears to be essential for proper condensation of the chromatin, although some controversy exists (Gusse et al., 1986
; Aoki and Carrell, 2003). A recent study evaluating the effects of organophosphorous pesticides on sperm protamine phosphorylation showed that the resulting sperm had abnormal chromatin condensation with subsequent DNA damage (Pina-Guzman et al., 2005). Abnormal phosphorylation may be relevant to human exposure to organophosphorous pesticides (Sanchez-Pena et al., 2004; Pina-Guzman et al., 2005, 2006).
The replacement of transition proteins with protamines induces a conformational change in the packaging of the chromatin. The chromatin forms loop domains, which are less than half the size of somatic cell histone loops, then forms toroidal structures, which have a 6–20-fold increase in packaging compaction (Ward and Coffey, 1991; Balhorn et al., 2000). The mechanism by which protamines induce the conformational changes is not well understood (D'Auria et al., 1993; Bianchi et al., 1994; Fuentes-Mascorro et al., 2000; Aoki and Carrell, 2003). P1 and P2 may bind to the major and minor groove of DNA or to the DNA surface by interacting electrostatically with phosphate residues (D'Auria et al., 1993; Bianchi et al., 1994; Balhorn et al., 1999; Fuentes-Mascorro et al., 2000).
Protamines are currently thought to be necessary for (i) condensing the male genome to generate a more compact and hydrodynamic nucleus, (ii) protecting the genetic message from nucleases, mutagens or damage from ROS or other factors, (iii) epigenetic modification during spermiogenesis and (iv) removing transcription factors and proteins to help reset the imprinting code in the oocyte (Oliva, 2006). Altering the sperm protamine content can disrupt any of the functions listed above.
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Regulation of protamine expression and potential causes of abnormalities |
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The human sperm haploid genome encodes a single copy of human P1 and P2 genes which maps to chromosome 16p13.3 (Domenjoud et al., 1991
). This locus also contains the TP2 gene. The P1–P2–TP2 locus spans a 28.5 kb region in which the three genes are arranged in a linear array, presumably facilitating concurrent or co-ordinated gene expression (Schluter et al., 1992; Choudhary et al., 1995). The P1 gene is present in all mammalian species, whereas P2 is present in mouse, hamster, rat, stallion and man (Calvin, 1976; McKay et al., 1985, 1986; Poccia, 1986; Bower et al., 1987; de Yebra et al., 1993). However, in some species, the protamine 2 gene is present but the protein is absent (Maier et al., 1990) . These findings suggest that P1 is inherited from a common ancestor, since it is present in all species and that the P2 gene may have been derived from the P1 gene through divergent evolution. Alternatively, the P1 and P2 genes may have been inherited from a single common ancestor, but successive species have lost the ability to express protamine 2 (Calvin, 1976; Oliva and Dixon, 1990). Several factors have been postulated and studied as possible causes of P1/P2 deregulation. These factors are summarized in Table I and discussed in the following sections.
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Gene polymorphisms |
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The protamine or transition protein genes could harbour mutations or polymorphisms that could induce conformational changes in the proteins, which could alter their incorporation into sperm chromatin. De Yebra et al. (1993)
performed a preliminary mutational analysis of the protamine gene analysis in four patients with markedly altered P1/P2 ratios with no mutations observed. Subsequently, Schlicker et al. (1994) screened 36 infertile patients with chromatin anomalies, but he failed to identify any mutations in the genes encoding P1, P2 or TP1. Tanaka et al. (2003) reported four synonymous polymorphisms in P1 and one SNP in P2 that generated a premature stop codon. The SNP in the P2 gene that induces translation termination may result in male infertility due to haploinsufficiency of P2.
Iguchi et al. (2006) sequenced the protamine genes in men exhibiting semen quality defects consistent with protamine abnormalities (i.e. sperm DNA damage). In their study, a heterozygous SNP which altered a highly conserved arginine residue was found in 10% (3/30) of the patients studied, but not seen in controls. This SNP converts one of the highly conserved arginines to a serine residue, therefore creating an RS sequence which can serve as a potential phosphorylation site for the enzyme SRPK1. Improper phosphorylation can substantially alter both DNA binding and protamine-to-protamine interaction in the sperm nucleus.
Recently, a larger patient population with known abnormal protamine ratios was screened to identify SNPs in the protamine and transition protein genes potentially responsible for the patients' altered protamine expression (Aoki et al., 2006a