Complete Selective Absence of Protamine P2 in Humans*

Male sterility due to abnormal sperm morphology or motion has been widely reported, although relatively little has been published on the sperm nuclear protein abnormalities. We report the first cases worldwide of infertile patients having a complete selective absence of protamine P2 in the sperm nucleus. This provides a selective phenotype that will aid understanding of the mechanisms of synthesis, processing, or function of the P2 protamines. In addition, it is of marked immediate relevance to medicine as it allows the diagnosis of this type of human male sterility and the opportunity to understand the basis of this defect.

The sperm nucleus is of special interest to researchers not only because it delivers the haploid genetic complement, but also because of its marked compositional and morphological changes as compared to the rest of the nucleus in the organism. The nucleohistone structure present in spermatocytes is disassembled during spermiogenesis in many species and replaced by a complex formed by the DNA condensed by protamines (for reviews see Mezquita (1985), Poccia (1986), Balhorn (1989), , Kasinsky (1989), and Oliva and Dixon (1991)). In humans, protamines are the most abundant nuclear proteins in the sperm nucleus packaging the male genome. There are two types of protamines: P1, which is present in all mammalian species studied so far and is rich in arginine and cysteine (Tobita et al., 1983;Sautiere et al., 1984;McKay et al., 1985;Lee et al., 1987;Domenjoud et al., 1990; Queralt and Oliva, 1991;Winkfein et al., 1991;Balhorn et al., 1992;Adroer et al., 1992;Retief et al., 1993), and the P2 family, composed of the P2, P3, and P4 components, which differ only by an amino-terminal extension of 1-4 residues' (McKay et al., 1986;Arkhis et al., 1991). Unlike P1 protamines, P2 protamines are only present in humans and a few other mammals (mouse, hamster, and stallion among others) and are in addition histidine-rich (McKay et al., 1986;Gusse et al., 1986;Bower et al., 1987;Lescoat et al., 1988;Sautihre et al., 1988;Bellv6 et al., 1988;Domenjoud et al., 1988Domenjoud et al., , 1991Lee et al., 1991;Arkhis et al., 1991; Oliva and Dixon, 1991;Chauvihre et al., 1992;Pirhonen et al., 1990). The functional and evolutionary differences between P1 and P2 protamines are not completely understood at present. In addition to having been found in all mammals studied, P1type protamines have also been found in birds ( , suggesting a more universal role of P1 protamines in the sperm nucleus than the P2 protamines, which are found only in some mammals. It has been demonstrated that bull, boar, and rat (which lack P2 in the sperm nucleus) still contain the P2 gene in their respective genomes (Tanhauser Bunick et al., 1990;Maier et al., 1990). The lack of P2 in boar and bull has been attributed to mutations in the genes and respective primary sequence of the protein, which could weaken the affinity to DNA (Maier et al., 1990). In the case of the rat, evidence for the presence of P2 precursor in the mature rat sperm has been presented (Stanker et al., 1992;Kremling et al., 1992). In humans, slight alterations in the P1/P2 ratio have been reported in different studies (Chevaillier et al., 1987;Balhorn et al., 1988;Bach et al., 1990;Blanchard et al., 1990;Belokopytova et al., 1993) in a few patients (2-17; altered P1/P2 ratio = 1.58 f 0.24, range 1.2-1.94; normal P1/P2 ratio = 0.98 f 0.12, range 0.79-1.27) (Balhorn et al., 1988). In this study we have analyzed the sperm samples from 116 random unselected patients from infertile couples. Because of the large number of samples analyzed, we have been able to detect new sperm nuclear compositional differences and trends among samples. One of the most remarkable findings has been the detection of a complete selective absence of protamine P2 in the sperm nuclei of 4 patients.

EXPERIMENTAL PROCEDURES
Unselected human sperm samples (ejaculates) from infertile couples attending the first visit to the infertility clinic were collected and kept frozen until processed. Approximately 14 X lo6 sperm cells from each sample were thawed and spun in 1.5-ml tubes in a microcentrifuge at 12.000 rpm at 4 "C for 5 min, the supernatant discarded, and the sediment washed twice (resuspending the cells by vortexing 10 s and centrifuging the cell suspension 5 min each time) with 200 GI of freshly prepared 1 mM phenylmethylsulfonyl fluoride (Sigma) in H20 (to lyse the cells by hypotonic shock). The sediment was then processed and analyzed by polyacrylamide gel electrophoresis as described (Yebra and Oliva, 1993). Polyacrylamide gel electrophoresis was performed on 23-cm gels containing 0.9 M acetic acid, 2.5 M urea, 15% acrylamide, 0.09% bisacrylamide, 0.12% ammonium persulfate, 0.4% TEMED.' The gels were stained with 1.1 g of Coomassie Blue R-250 (Bio-Rad) dissolved in 250 ml of methanol, 250 ml of HzO, 50 ml of acetic acid during 45 min, destained 5 min in 50% methanol, 10% acetic acid, and destained overnight in 10% methanol, 10% acetic acid (Fig. 1). The gels were then dried between two sheets of cellophane film and the intensity of the bands quantified by scanning each lane (Hoeffer Scientific linear scanner) and subsequently meas-The abbreviation used is: TEMED, N,N,N',N'-tetramethylethylenediamine.

Protamine P2 Absence in Humans
uring the area of each peak with a digitizing tablet (Kontron). The ratio P1/P2 was then calculated, and the samples were ordered and plotted according to decreasing P1/P2 ratios ( Fig. 2.4). The ratio to total nuclear protein of the intermediate proteins ( Fig. 2B), proteins with a migration similar to histones (Fig. 2C), and total protamines ( Fig. 2 0 ) were also calculated and expressed in function of samples ordered in decreasing P1/P2 ratios. The ratios P1/P2 for each sample was also plotted as a function of the different parameters studied in order to further demonstrate correlations among parameters (Fig. 3).

RESULTS
Analysis of the sperm nuclear proteins from 116 different individuals revealed that 22.4% of these had a normal P1/P2 ratio (1.10 & 0.08, range 0.92-1.27) and 74.1% had an altered P1/P2 ratio (3.00 f 2.84, range 1.28-15.52). Unexpectedly, four (3.4%) samples had a complete lack of P2 protamine in their sperm nucleus, whereas their protamine P1 levels were conserved ( Fig. 1). Sperm count corresponding to the samples with a complete selective absence of P2 was 10.6 f 7.1 x lo6 sperm cells/ml, the ejaculate volume was 3.8 f 1.4 ml, abnormal sperm cells were 63.5 f 11.4%, and astenozoospermia was present (progressive motility was 3.3%, range 0-13.3%; difficult progressive motility 18.4 & 9.1%; and non-progressive motility was 0.8%, range 0-3.3%). Because of the large number of samples analyzed, many new trends have also emerged (Figs. 2 and 3). 1) Samples with a higher P1/P2 ratio (lower P2) also contained a higher proportion of intermediate proteins (see Figs. 2B and 3B). 2) The proteins with a migration similar to that of histone standards covered a wide range of levels (Figs. 2C and 3C). A large proportion of the samples with an altered P1/P2 ratio also had an increased level of proteins with a mobility similar to histones (Fig. 3C). 3) Samples with an increased P1/P2 ratio (lower P2) also had decreased total protamine levels (Figs. 2 0 and 3A). 4) Samples with a low sperm count (below 25 million/ml) were also more likely to have an altered P1/P2 ratio (Fig. 3 0 ) .
With the above data, it is now possible to propose a more detailed classification of the human sperm nuclear protein ratios as follows: 1) complete selective absence of P2 (3.4%),  Yebra and Oliva, 1993). Lune I , standard of chicken erythrocyte histones; lane 2, infertile patient with a complete absence of protamine P2; lunes 3 and 4, infertile patients with altered and normal P1/P2 ratios, respectively. INTERMEDIATE PROTEINS refers to human transition proteins 1 and 2 as well as P2 family precursors (HPS1 and HPS2) at early stages of processing among others (Gusse et al., 1986;Sautibre et al., 1988;Luerssen et al., 1990;Schluter et al., 1992). PI, protamine P1. P2 family, protamines P2, P3, and P4, which have been sequenced and differ only by an amino acid extension of 1-4 residues (Arkhis et al., 1991). 2) altered P1/P2 ratio with increased levels of intermediate proteins above 0.3 with respect to total nuclear protein (61.2%), 3) altered P1/P2 ratio with levels of intermediate proteins below 0.3 respect to total nuclear protein (12.9%), 4) normal P1/P2 ratio but increased levels of intermediate proteins over 0.3 with respect to total nuclear protein (11.2%), 5) normal P1/P2 ratio with levels of intermediate proteins below 0.3 with respect to total nuclear protein (11.2%). In 1 of these 4 patients, an independent semen sample obtained 6 months later also reproducibly demonstrated the complete selective absence of P 2 family proteins. This indicates that this phenotype was present in this patient a t least during this period of time.

DISCUSSION
In this paper we have analyzed the nuclear proteins of 125 sperm samples from different human individuals and detected the first examples worldwide of a complete selective absence of protamine P2 in the human sperm nucleus as well as many different new trends in the ratio of sperm nuclear proteins. This is the most extensive study (116 different patients plus 9 controls) performed so far analyzing the human sperm nuclear proteins. The absence of identification of this novel type of alteration by previous authors most likely reflects the small number of infertile patients previously studied by them (between 2 and 17 at most), although epidemiological or ethnical differences could also be present. The much larger proportion of patients having only a decrease in the levels of P2 compared with the small number of patients having a complete selective absence of P2 suggests that the most frequent type of alteration could be a heterozygous form of a genetic defect causing the complete selective absence of protamine P 2 when in a homozygous state. Several families with multiple infertile males have been described (Cantu et al., 1981;Chaganti et al., 1980;Florke-Gerloff et al., 1984). The average number of years of sterility history in the selective P 2 absence patients is 6.4 f 0.3. None of these patients has ever been a father.
The detection of this selective absence of P2 protamine in the sperm nucleus offers a unique opportunity to determine the mechanisms leading to this type of sterility. For instance, the complete absence could arise either from a lack of expression of the P2 gene or from anomalous processing of the P2 precursor and subsequent transport into the nucleus and binding to the DNA. In the boar, it has been shown that the lack of P2 in the sperm nucleus is due to mutations within the P 2 gene (Maier et al., 1990). We have sequenced the protamine genes from these patients in search for mutations. Preliminary results indicate that no mutations are present at the coding region level or in the promoter up to -200 base pairs from the transcriptional start site in any of these four patients. In order to narrow down the search for the primary defect, we have also initiated the study of the expression of the protamine genes in testicular biopsy samples in search for altered expression or processing of the P2 precursor. It is not clear whether the lack of P2 in this patients is the primary defect leading to sterility or whether it merely represents an indirect factor contributing to sterility due to a different primary cause. Many factors that impair spermatogenesis (toxic agents, deficiencies, infections, inmunological changes, and hormonal changes) could lead to oligospermia and/or decreased P2 levels (Aiman et al., 1979;Snyder, 1990;Ueda et al., 1991;Fraga et al., 1991;J6gou et al., 1991;Pavlou et al., 1991;van Beek and Meistrich, 1992). It should also be considered a possibility that several independent causes (for example different diseases) affecting spermatogenesis could all lead through independent mechanisms to a common alteration in the levels of P2 proteins, ranging from slight changes in the P1/P2 ratio to the complete selective absence of P2.
Decreased levels of P2 in the nucleus could originate in the final stages of spermiogenesis before the synthesis of P2 has been completed or processed. In the mouse it has been shown that the initiation of the synthesis of the "tyrosine variants of protamine" (Pl) must be separated by at least 1 day by the synthesis of the "histidine variants of protamine" (P2) (Balhorn et al., 1984). An uncoupling in the generation of mature forms of P1 and P2 protamines could be originated by the fact that P2s are synthesized as precursors that must be processed, whereas Pls are not. A general failure of the processing of proteins (including P2s) or of the nucleohistone to nucleoprotamine replacement mechanisms (Oliva et al., 1987(Oliva et al., , 1990; Oliva and Dixon, 1991) at this level, which could be caused by different defects or agents (see above), should result in a relative decrease or the absence of P2 protamines in the gametic cells found in the semen. The presence of increased levels of intermediate proteins3 in samples with lower P1/P2 ratios (Figs. 2B and 3B) may indicate incomplete processing of P2 proteins.
In addition to the immediate relevance to medicine, the findings reported here also represent a unique opportunity to understand many basic aspects of the expression, control, and function of the protamines. The fact that all of the P2 family components have disappeared could support the idea that all P2, P3, and P4 are derived from a single gene (Arkhis et al., 1991). Balhorn et al. (1988) have reported that the levels of P2 decrease in infertile patients, while the levels of P3 are indistinguishable from that of normal samples. Our results indicate that the ratio P2/P3 (control = 2.48 f 0.54; infertile group = 2.73 f 0.68; Fig. 2E) is comparable to that detected by Balhorn et al. (1988) (control group = 3.17 +_ 0.68; infertile group = 2.38 f 0.25), although we have not detected a significant correlation between altered Pl/PP-farnily ratios and the ratio P2/P3 after analyzing samples from 116 different patients (Fig. 2E). Belokopytova et al. (1993) suggest that in normal sperm P2s bind more strongly to DNA than Pls but that the affinity of P2s to DNA in infertile patients has been reduced. Since we have not detected any mutations at the coding region level of the protamine P2 genes in any of these four patients lacking P2, the hypothesis of mutations causing decreased affinity of mutated P2s to DNA can be discarded in these cases. Despite the extremely positively charged nature of the protamines and the obvious participation in condensing the DNA in the sperm nucleus, the basic function of the protamines remains unknown at present ( Oliva and Dixon, 1991). The basic hypotheses are: 1) condensation and stream- lining of the sperm nucleus, 2) protection of the genetic message delivered by the spermatozoa, and 3) generation of an imprintedhlank state in the male genome ( Oliva and Dixon, 1991). It is interesting to note that the P2 protamines are zinc finger proteins with a Cys-2/His-2 motif (Bianchi et al., 1992), which is a motif shared by many transcription factors.
The complete selective absence of P2 in these patients should lead to a loss of the structural, biochemical, and functional aspects normally provided by this protamine. In this sense we have initiated the study of the sperm morphological nuclear changes of samples having a complete selective absence of protamine P2 as compared to normal sperm containing the full complement of P1 and P2 protamines. Preliminary results indicate a marked decondensation of the P2 lacking nuclei as compared to the samples having a full complement of P1 and P2 protamines. This marked decondensation is not necessarily due to the lack of P2, but could also be produced by an increased proportion of intermediate proteins or of proteins migrating at the level of histones. The simple overall reduction in the levels of total protamine (due to the decrease in P2), which is not compensated by an increase of P1, could also contribute to the lack of condensation. Decondensed or unstable sperm nuclei in unfertile patients have been reported (Evenson et al., 1980;Bustos-Obreg6n and Leiva, 1983;Colleu et al., 1988;Dadoune et al., 1988;Blanchard et al., 1990;Jager, 1990;Auger, 1990;Pasteur et al., 1992). It will be interesting to determine whether there is a correlation or coincidence of this type of phenotype (selective absence of protamine P2 family) with previously described types of sperm based on morphological, dynamic, clinical, or cytogenetic considerations (such as globozoospermia, spermatid arrest, or chromosomal aberrations) (Navarro et al., 1990;De Braekeleer and Dao, 1991;Bibbins et al., 1992;Meyer et al., 1992). It will be equally interesting to determine if the physiological decondensation of the sperm nucleus occurring post-fertilization in the egg is altered in nuclei selectively lacking P2 by mimicking the decondensation process in vitro through incubation of the sperm cells with egg extracts or other agents (Huret, 1986;Brown et al., 1992;Lohka and Masui, 1983).